Method for mass humanization of non-human antibodies

ABSTRACT

The present invention relates to a method for producing a population of nucleic acids encoding at least one protein comprising at least one immunoglobulin variable domain having a non-human-derived CDR3 amino acid sequence embedded in essentially human framework sequences, as well as to a population of nucleic acids and a population of proteins relates thereto and uses thereof.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 16/156,740, filed Oct. 10, 2018, which is adivisional application of U.S. patent application Ser. No. 15/130,843,filed Apr. 15, 2016, now U.S. Pat. No. 10,125,198, which claims thebenefit of U.S. Provisional Application No. 62/155,421, filed Apr. 30,2015 and U.S. Provisional Application No. 62/149,440, filed Apr. 17,2015, all of which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 16, 2021, isnamed 60864-701_301_SL.txt and is 1,202 bytes in size.

BACKGROUND OF THE INVENTION

Natural immunoglobulins have been used in assays, diagnosis and, to amore limited extent, therapy. However, such uses, especially in therapy,have been hindered by the polyclonal nature of natural immunoglobulins.The advent of monoclonal antibodies of defined specificity increased theopportunities for therapeutic use. However, most monoclonal antibodiesare produced following immunization of a rodent host animal with thetarget protein, and subsequent fusion of a rodent spleen cell producingthe antibody of interest with a rodent myeloma cell. They are,therefore, essentially rodent proteins and as such are naturallyimmunogenic in humans, frequently giving rise to an undesirable immuneresponse termed the HAMA (Human Anti-Mouse Antibody) response.

Previous attempts to decrease the immunogenicity of therapeuticantibodies have traditionally used a human template that is selected bythe degree of homology to the donor antibody (the human antibody mosthomologous to the non-human antibody in the variable region is used asthe template for humanization). Although this approach has been shown towork, it limits the possibility of selecting the best human templatesupporting the donor CDRs. Moreover, a CDR grafted humanized antibodyprepared in this way may demonstrate a significantly decreased bindingaffinity.

SUMMARY OF THE INVENTION

In view of the foregoing, there is a need for methods and librarieswhich allow for quick and/or effective humanization of antibodies, inparticular for mass humanization of antibodies. There is further a needfor methods and libraries which allow for identification of humanizedantibodies binding to a target of interest in only one selection roundand/or without the need for cloning immunoglobulin domains intodifferent vectors during selection. The methods and compositionsdescribed herein address these needs and provided additional advantagesas well.

In one aspect, the disclosure provides a method of producing apopulation of nucleic acids encoding at least one protein comprising atleast one immunoglobulin variable domain having a non-human-derived CDR3amino acid sequence embedded in essentially human framework sequences.In one embodiment, the method comprises the steps of: (a) providing atleast one nucleic acid encoding a non-human-derived complementaritydetermining region 3 (CDR3) amino acid sequence or an amino acidsequence further encompassing 1, 2, or 3 amino acids N-terminal and/orC-terminal of the non-human-derived CDR3 amino acid sequence; (b)generating a population of nucleic acids encoding at least one proteincomprising at least one immunoglobulin variable domain having anon-human CDR3 amino acid sequence of step (a) embedded in essentiallyhuman framework sequences, wherein the human framework sequencescomprise a first human framework region (FR1), a second human frameworkregion (FR2), a third human framework region (FR3), and a fourth humanframework region (FR4), such that the FR1 and FR2 regions areinterspaced by a complementarity determining region 1 (CDR1), the FR2and FR3 regions are interspaced by a complementarity determining region2 (CDR2), and the FR3 and FR4 regions are interspaced by anon-human-derived CDR3 amino acid sequence; wherein the nucleic acidsequences encoding the CDR1 and CDR2 amino acid sequences arediversified among the population of nucleic acids encoding at least oneprotein comprising at least one immunoglobulin variable domain; whereineach nucleic acid sequence encoding a CDR1 or CDR2 amino acid sequenceis independently based (i) on a nucleic acid sequence encoding a humanCDR1 or CDR2, respectively, or (ii) on a nucleic acid sequence encodinga non-human CDR1 or CDR2, respectively; wherein at least some of thenucleic acid sequences encoding a CDR1 or CDR2 amino acid sequence havebeen modified to encode at least one amino acid present in non-humanCDR1 or CDR2 amino acid sequences, respectively, in case of human CDR1or CDR2, respectively, or to encode at least one amino acid present inhuman CDR1 or CDR2 amino acid sequences, respectively, in case ofnon-human CDR1 or CDR2, respectively; and wherein the human FR1, FR2,FR3 and FR4 regions are human framework regions selected to provide ascaffold conducive for non-human CDR3 amino acid sequences, with theproviso: that the two C-terminal amino acids of FR2 are optionallynon-human, and that the two C-terminal amino acids of FR3 are optionallynon-human.

In another aspect, the disclosure provides a method of producing apopulation of nucleic acid sequences encoding at least one proteincomprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences. In some embodiments, the method comprises one ormore steps of a process as described herein.

In another aspect, the disclosure provides a method of producing apopulation of amino acid sequences for one or more proteins comprisingat least one immunoglobulin variable domain having a non-human-derivedCDR3 amino acid sequence embedded in essentially human frameworksequences. In some embodiments, the method comprises one or more stepsof a process as described herein.

In one aspect, the disclosure provides a population of nucleic acids inaccordance with any of the methods described herein.

In one aspect, the disclosure provides a population of proteins inaccordance with any of the methods described herein.

In one aspect, the disclosure provides a system for performing any ofthe methods described herein. In some embodiments, the system comprisesa computer processor programmed to perform one or more steps of themethod.

In one aspect, the disclosure provides computer readable mediumcomprising machine executable code that upon execution by one or morecomputer processors implements any of the methods described herein.

In one aspect, the disclosure provides uses of the various compositionsdescribed herein with regard to any of the various aspects. In someembodiments, a composition of the disclosure is used in the preparationof a medicament for the treatment of a disease or condition of asubject.

In one aspect, the disclosure provides a method of treating a subjectfor a disease or condition. In some embodiments, the method comprisesadministering to a subject one or more proteins of any of the variousaspects disclosed herein.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification, and applications to which the present application claimsthe benefit of priority are herein incorporated by reference to the sameextent as if each individual publication, patent, or patent applicationwas specifically and individually indicated to be incorporated byreference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows the structure of light and heavy chain immunoglobulinframework regions and CDRs.

FIG. 2 shows how overlap PCR can be used to generate humanized acceptorlibraries containing non-human CDR3 sequences.

FIG. 3 shows exemplary positional weight matrices (PWM) for human lightchain V segment IGHV1-3 CDRS.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, various embodiments may be presented in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation. Accordingly, the description of a rangeshould be considered to have specifically disclosed all the possiblesubranges as well as individual numerical values within that range. Forexample, description of a range such as from 1 to 6 should be consideredto have specifically disclosed subranges such as from 1 to 3, from 1 to4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.This applies regardless of the breadth of the range.

The systems and methods of this disclosure as described herein mayemploy, unless otherwise indicated, conventional techniques anddescriptions of molecular biology (including recombinant techniques),cell biology, biochemistry, microarray and sequencing technology, whichare within the skill of those who practice in the art. Such conventionaltechniques include polymer array synthesis, hybridization and ligationof oligonucleotides, sequencing of oligonucleotides, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, N.Y. (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3rd Ed., W.H. Freeman Pub., New York (2000);and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub., New York(2002), all of which are herein incorporated by reference in theirentirety for all purposes. Before the present compositions, researchtools and systems and methods are described, it is to be understood thatthis disclosure is not limited to the specific systems and methods,compositions, targets and uses described, as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended tolimit the scope of the present disclosure, which will be limited only byappended claims.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA(rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide may comprise one or more modified nucleotides,such as methylated nucleotides and nucleotide analogs. If present,modifications to the nucleotide structure may be imparted before orafter assembly of the polymer. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions. Sequenceidentity, such as for the purpose of assessing percent complementarity,may be measured by any suitable alignment algorithm, including but notlimited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needlealigner available at ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html,optionally with default settings), the BLAST algorithm (see e.g., theBLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi,optionally with default settings), or the Smith-Waterman algorithm (seee.g., the EMBOSS Water aligner available atebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally withdefault settings). Optimal alignment may be assessed using any suitableparameters of a chosen algorithm, including default parameters.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 Daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Chothia et al., J.Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.U.S.A. 82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see e.g. Kabat etal., Sequences of Proteins of Immunological Interest, Fifth Edition,National Institute of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily.Pepsin treatment yields an F(ab′)2 fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.The Fab fragment also contains the constant domain of the light chainand the first constant domain (CHI) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CHI domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known. The“light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these can be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

CDR-1H, CDR-2H, and CDR-3H denote immunoglobulin heavy chaincomplementarity determining region 1, 2 and 3 respectively. VHFR1,VHFR2, and VHFR3 VHFR4 denote immunoglobulin heavy chain frameworkregion 1, 2, 3 and 4 respectively. CDR-1L, CDR-2L, and CDR-3L denoteimmunoglobulin light chain complementarity determining region 1, 2 and 3respectively. VLFR1, VLFR2, and VLFR3 VLFR4 denote immunoglobulin lightchain framework region 1, 2, 3 and 4 respectively.

The heavy/light (H/L) interface mount angle is measured as the degreeshift of the central axis of the light chain Fv compared to a fixedsuperposition of heavy chain Fv in a predicted or observed crystalstructures. Non-limiting exemplary methods of determining the H/Linterface mount angle, alternately referred to as the packing angle, canbe found in Dunbar et al. ABangle: characterizing the VH-VL orientationin antibodies. Protein Engineering, Design, and Selection 26, 611-620(2013).

A positional weight matrix can be a matrix indicating, for each of aplurality of positions within a polypeptide, the relative frequency of agiven amino acid within a population of variants of the polypeptide. Forexample, if a multiple protein alignment indicates that three variantshave an S, R, and a T at a given position in an alignment, then withinthe positional weight matrix at that position would be [S:0.33, R:0.33,T:0.33] indicating the identity and relative frequency of the aminoacid.

In one aspect, the disclosure provides a method for producing apopulation of nucleic acids encoding at least one protein comprising atleast one immunoglobulin variable domain having a non-human-derived CDR3amino acid sequence embedded in essentially human framework sequences,as well as to a population of nucleic acids and a population of proteinsrelates thereto and uses thereof.

In one aspect, provided herein is a method for producing a population ofnucleic acids encoding at least one protein comprising at least oneimmunoglobulin variable domain having a non-human-derived CDR3 aminoacid sequence embedded in essentially human framework sequences. Themethod can comprise the following steps: (a) providing at least onenucleic acid encoding a non-human-derived complementarity determiningregion 3 (CDR3) amino acid sequence or an amino acid sequence furtherencompassing 1, 2, or 3 amino acids N-terminal and/or C-terminal of thenon-human-derived CDR3 amino acid sequence and (b) generating apopulation of nucleic acids encoding at least one protein comprising atleast one immunoglobulin variable domain having a non-human CDR3 aminoacid sequence of step (a) embedded in essentially human frameworksequences. The human framework sequences can comprise a first humanframework region (FR1), a second human framework region (FR2), a thirdhuman framework region (FR3), and a fourth human framework region (FR4),such that the FR1 and FR2 regions are interspaced by a complementaritydetermining region 1 (CDR1), the FR2 and FR3 regions are interspaced bya complementarity determining region 2 (CDR2), and the FR3 and FR4regions are interspaced by a non-human-derived CDR3 amino acid sequence.The nucleic acid sequences encoding the CDR1 and CDR2 amino acidsequences can be diversified among the population of nucleic acidsencoding at least one protein comprising at least one immunoglobulinvariable domain, wherein each nucleic acid sequence encoding a CDR1 orCDR2 amino acid sequence is independently based (i) on a nucleic acidsequence encoding a human CDR1 or CDR2, respectively, or (ii) on anucleic acid sequence encoding a non-human CDR1 or CDR2, respectively,wherein at least some of the nucleic acid sequences encoding a CDR1 orCDR2 amino acid sequence have been modified to encode at least one aminoacid present in non-human species CDR1 or CDR2 amino acid sequences,respectively, in case of human CDR1 or CDR2, respectively, or to encodeat least one amino acid present in human CDR1 or CDR2 amino acidsequences, respectively, in case of non-human species CDR1 or CDR2,respectively. The human FR1, FR2, FR3 and FR4 regions can be humanframework regions selected to provide a scaffold conducive for non-humanCDR3 amino acid sequences, with the proviso that the two C-terminalamino acids of FR2 are optionally non-human, and that the two C-terminalamino acids of FR3 are optionally non-human.

Non-limiting examples of non-human species contemplated herein includemouse, dog, cats, zebrafish, llamas, shark, cow, and rats. In oneinstance a non-human species can be selected from the group consistingof mouse, rat, dog, cat, cow, sheep, pig, horse, donkey, goat, chicken,ferret, pika, bat, llama, bear, tiger, wolf, fox, lion, cheetah,giraffe, African wild dog, monkey, ape, orangutan, chimpanzee, rhesusmonkey, macaque, squirrel, lizard, snake, alligator, turtle, crocodile,tortoise, toad, frog, newt, salamander, duck, goose, guinea fowl, guineapig, hamster, penguin, ostrich, quail, turkey, owl, scrub jay,zebrafish, catfish, eel, shark, swordfish, Antarctic fish (TrBel),Antarctic fish (GyAcl), white sturgeon, or any other animal thatproduces immunoglobulins.

“A population of nucleic acids” is understood as 2 or more nucleicacids, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 or morenucleic acids, wherein at least 2 of the nucleic acids of the populationexhibit different nucleic sequences, more preferably wherein at least 2,3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 or more nucleic acids of thepopulation exhibit different nucleic sequences, and/or wherein at least50%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% of the nucleic acids of thepopulation exhibit different nucleic sequences.

In some embodiments, an encoded protein comprises at least oneimmunoglobulin variable domain, preferably 1, 2, 3, 4 or moreimmunoglobulin variable domains, more preferably 1 or 2 immunoglobulinvariable domains. For example, a variable heavy immunoglobulin domainmay be paired with a variable light domain to provide an antigen bindingsite; such as in an scFv. Alternatively, independent regions, e.g., avariable heavy domain alone or a variable light domain alone may beused. An immunoglobulin variable domain comprises CDR1, CDR2 and CDR3sequences. In particular, an immunoglobulin variable heavy domaincomprises CDR-1H, CDR-2H and CDR-3H sequences, and an immunoglobulinvariable light domain comprises CDR-1L, CDR-2L and CDR-3L sequences.

A “non-human CDR3 amino acid sequence” is understood as an amino acidsequence which is identical to a CDR3 amino sequence naturally occurringin a non-human antibody. The CDR3 amino sequence may be a CDR-3L or aCDR-3H amino acid sequence.

A “non-human-derived CDR3 amino acid sequence” is understood as an aminoacid sequence which is identical to a CDR3 amino sequence naturallyoccurring in a non-human antibody, or which contains 1, 2, 3, 4, or 5amino acid mutations compared to a CDR3 amino sequence naturallyoccurring in a non-human antibody, preferably wherein the mutation is aconservative mutation.

Conservative amino acid substitutions, as one of ordinary skill in theart will appreciate, are substitutions that replace an amino acidresidue with one imparting similar or better (for the intended purpose)functional and/or chemical characteristics. For example, conservativeamino acid substitutions are often ones in which the amino acid residueis replaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). The purpose formaking a substitution is not significant and can include, but is by nomeans limited to, replacing a residue with one better able to maintainor enhance the structure of the molecule, the charge or hydrophobicityof the molecule, or the size of the molecule. For instance, one maydesire simply to substitute a less desired residue with one of the samepolarity or charge. Such modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. One specific means by which those of skill inthe art accomplish conservative amino acid substitutions is alaninescanning mutagenesis. The altered polypeptides are then tested forretained or better function using functional assays available in theart.

Nucleic acid molecules may be in the form of RNA, such as mRNA or cRNA,or in the form of DNA, including, for instance, cDNA and genomic DNAe.g. obtained by cloning or produced by chemical synthetic techniques orby a combination thereof. The DNA may be triple-stranded,double-stranded or single-stranded. Single-stranded DNA may be thecoding strand, also known as the sense strand, or it may be thenon-coding strand, also referred to as the anti-sense strand. Nucleicacid molecule as used herein also refers to, among other, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, nucleic acid molecule as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA.

The nucleic acids may be originally formed in vitro or in a cell inculture, in general, by the manipulation of nucleic acids byendonucleases and/or exonucleases and/or polymerases and/or ligasesand/or recombinases or other methods known to the skilled practitionerto produce the nucleic acids.

The term “embedded in essentially human framework sequences” isunderstood as that the CDR3-derived sequence is located within theframework sequences to yield an immunoglobulin variable domain. Forexample, a skilled person is aware that a CDR-3L amino acid sequence islocated between FR3 and FR4 framework regions of the light chain in caseof an immunoglobulin light chain variable domain.

“Human framework sequences” are understood as framework sequences whichare naturally occurring human framework sequences. The nucleic acidsencoding the human framework sequences may contain silent mutations ascompared to the naturally occurring nucleic acids encoding the humanframework sequences and/or sequences that are a result of thedegeneration of the genetic code. There are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all nucleotidesequences are included which result in the human framework sequences asdefined above.

An “essentially human framework sequence” is understood as frameworksequence which exhibits at least 90%, preferably at least 95%, 96%, 97%,98%, or 99% sequence identity to a naturally occurring human frameworksequence. In a preferred embodiment, the essentially human frameworksequence consists of FR1, FR2, FR3 and FR4 regions, which are human FR1,FR2, FR3 and FR4 regions, with the proviso that the two C-terminal aminoacids of FR2 are optionally non-human, and that the two C-terminal aminoacids of FR3 are optionally non-human, more preferably, the twoC-terminal amino acids of heavy FR2 are optionally non-human, and thatthe two C-terminal amino acids of heavy FR3 are optionally non-human.

“An amino acid sequence further encompassing 1, 2, or 3 amino acidsN-terminal and/or C-terminal of the non-human-derived CDR3 amino acidsequence” is understood as that the non-human-derived CDR3 amino acidsequence further comprises 1, 2, or 3 amino acids N-terminal of thenon-human-derived CDR3 amino acid sequence, and/or 1, 2, or 3 aminoacids C-terminal of the non-human-derived CDR3 amino acid sequence.

A “non-human-derived amino acid sequence” is understood as an amino acidsequence which is identical to a amino sequence naturally according in anon-human antibody, or which contains 1, 2, 3, 4, or 5 amino acidmutations compared to an amino sequence naturally occurring in anon-human species antibody, preferably wherein the mutation is aconservative mutation.

In some embodiments, at least one nucleic acid encoding anon-human-derived complementarity determining region 3 (CDR3) amino acidsequence or an amino acid sequence further encompassing 1, 2, or 3 aminoacids N-terminal and/or C-terminal of the non-human species-derived CDR3amino acid sequence is provided. In a preferred embodiment, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 50, 100 or more nucleic acids as definedabove are provided. In general, the methods and populations of thedisclosure are suitable for mass hybridization of the non-humanantibodies, and for providing a mass humanized library suitable for thispurpose. Therefore, it is preferred that more than one, preferably 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100 or more nucleic acids as definedabove are provided.

For efficient mass hybridization, it is preferred that the same methodis used for transferring the non-human or non-human-derived CDR3 regionsinto an Acceptor Framework of the disclosure, to obtain the populationof nucleic acids of step (b), which preferably represents nucleic acidsencoding a humanized library of antibodies. Therefore, it is preferredto provide either always CDR3 regions of the non-human antibodies, oralways a CDR3 region which further encompasses 1, 2, or 3 amino acidsN-terminal and/or C-terminal of the non-human-derived CDR3 amino acidsequence.

Therefore, in a further preferred embodiment, more than one, preferably2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100 or more nucleic acids asdefined above are provided, wherein the each nucleic acid encodes anon-human-derived complementarity determining region 3 (CDR3) amino acidsequence, preferably a mouse complementarity determining region 3 (CDR3)amino acid sequence.

In a yet further preferred embodiment, more than one, preferably 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100 or more nucleic acids as definedabove are provided, wherein the each nucleic acid encodes anon-human-derived complementarity determining region 3 (CDR3) amino acidsequence, preferably a non-human complementarity determining region 3(CDR3) amino acid sequence, which further encompasses 1, 2, or 3 aminoacids N-terminal and/or C-terminal of the non-human-derived CDR3 aminoacid sequence.

The phrase “interspaced by” in the context of the methods of thedisclosure is understood that two amino acid sequences are connected viathe interspacing amino acid sequence, preferably by peptide linkages.For example, a protein comprising the structure FR1-CDR1-FR2 isunderstood as that FR1 and FR2 regions are interspaced by a CDR1.

“CDR1 and CDR2 amino acid sequences are diversified among thepopulation” is understood as that at least 2 of the nucleic acids of thepopulation exhibit different CDR1 nucleic sequences, in particulardifferent CDR-1H and/or CDR-1L sequences, and/or at least 2 of thenucleic acids of the population exhibit different CDR2 nucleicsequences, in particular different CDR-2H and/or CDR-2L sequences, morepreferably wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150,200 or more nucleic acids of the population exhibit different CDR1and/or CDR2 nucleic sequences, and/or wherein at least 50%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% of the nucleic acids of the population exhibitdifferent CDR1 and/or CDR2 nucleic sequences.

In some embodiments, each nucleic acid sequence encoding a CDR1 or CDR2amino acid sequence is independently based (i) on a nucleic acidsequence encoding a human CDR1 or CDR2, respectively, or (ii) on anucleic acid sequence encoding a non-human (e.g., mouse) CDR1 or CDR2,respectively. “Based on” is understood as that, in case of a CDR1sequence, the CDR1 amino acid sequence contains at least 3, 4, 5, 6, 7,8, 9 or more, for example all, amino acids of a human CDR1, in the caseof (i), or of a non-human CDR1, in case of (ii), respectively, and/orexhibits at least 30%, at least 50%, at least 80%, at least 90%, atleast 95%, or 100% sequences identity to a human CDR1 (in case of i) orto a non-human CDR1 (in case of ii), respectively.

It is understood that the sequence comparison applies to the respectiveCDR1 regions; e.g. a CDR-1H region based on a human CDR-1H is comparedto human CDR-1H regions.

A “scaffold conducive for non-human CDR3 amino acid sequences” isunderstood as a immunoglobulin scaffold which is suitable for asuccessful graft of a non-human CDR3 amino acid sequence to yield acorrectly folded antibody for at least 30%, at least 50%, or at least60%, 70% or 80% of grafted non-human CDR3 amino acid sequences, and/orwhich exhibits at least 30%, at least 50%, at least 80%, or at least90%, framework homology to a non-human framework, and/or which exhibitsCDR cosmology, and/or wherein the CDR sequences exhibit canonicalstructures, and/or wherein the grafted non-human CDR3 sequences canadopt comfortable heavy or light mount angles, respectively. Therationale is that the framework scaffold serves to hold thenon-human-derived CDR3 sequences in their correct spatial orientationfor interaction with an antigen. Thus, if the selected essentially humanframework sequences selected to be similar to the non-human frameworks,it will maximize the likelihood that affinity will be retained in themass humanized antibodies.

Determining CDR regions and framework regions can be performed bymethods known in the art, as for example described in the chapterProtein Sequence and Structure Analysis of Antibody Variable Domains(in: Antibody Engineering Lab Manual, 2001 (Ed.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg).

For example, the CDR regions can be determined using the Kabatnomenclature, as described in Kabat et al. (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)), the Chothia nomenclature,as for example described in Al-Lazikani et al., ((1997) JMB273,927-948), the Martin nomenclature or the Contact nomenclature, asdescribed in MacCallum, R. M., Martin, A. C. R. and Thornton, J. T.(1996; Antibody-antigen interactions: Contact analysis and binding sitetopography. J. Mol. Biol. 262, 732-745).

TABLE 1 An overview on the location of a CDR region. Loop Kabat ChothiaContact L1 L24-L34 L24-L34 L30-L36 L2 L50-L56 L50-L56 L46-L55 L3 L89-L97L89-L97 L89-L96 H1 H31-H35B H26-H32 . . . 34 H30-H35B (Kabat Numbering)H1 H31-H35  H26-H32  H30-H35  (Chothia Numbering) H2 H50-H65  H52-H56 H47-H58  H3 H95-H102 H95-H102 H93-H101

In a preferred embodiment, the Kabat nomenclature is applied.

CDR-H3 has a length of 3 to 25 amino acids, and preferably starts 33residues after the end of CDR-H2 and generally 2 after a Cys. Residuesbefore CDR-H3 are preferably Cys-XXX-XXX, and are typically Cys-Ala-Arg.Residues after CDR-H3 are preferably Trp-Gly-XXX-Gly.

By the populations and methods of the disclosure, general solutions to anon-human antibody repertoire is provided, instead of providing singlesolutions to a single antibody example, thereby allowing masshumanization of a set of non-human antibodies and providing humanizedantibodies binding to a target of interest specifically and/or with highaffinity, even after a single selection round.

The human FR1, FR2, FR3 and FR4 regions are human framework regions withthe proviso that the two C-terminal amino acids of FR2 are optionallynon-human, and that the two C-terminal amino acids of FR3 are optionallynon-human.

Human FR1, FR2, FR3 and FR4 regions are FR1, FR2, FR3 and FR4 regionswhich are naturally occurring in humans. As described above, the twoC-terminal amino acids of FR2 are optionally non-human, and that the twoC-terminal amino acids of FR3 are optionally non-human. The FR1 and FR4regions can be human FR1 and FR4 regions. As also described above, it ispossible that a nucleic acid encoding a non-human-derived CDR3 aminoacid sequence, preferably a non-human-CDR3 amino acid sequence, furthercomprising 1, 2, or 3 amino acids N-terminal of the non-human-derivedCDR3 amino acid sequence, and/or 1, 2, or 3 amino acids C-terminal ofthe non-human-derived CDR3 amino acid sequence is provided, inparticular in case a nucleic acid encoding a non-human specificitydetermining region (SDR) as described above is provided. For example, anSDR comprising the CDR-3H may be grafted.

Further, FR2 is a human FR2 framework region, or the two C-terminalamino acids of FR2 are optionally non-human.

In one preferred embodiment, FR2 is a human FR2 framework region.

Various methods are available for providing a population of nucleicacids according to step (b) of the above method, starting from the atleast one nucleic acids of step (a).

For example, it is possible to generate a set of oligonucleotidescomprising sequences encoding non-human CDR3 regions and add those viaPCR to pre-amplified VH and VL libraries with the features of thedisclosure.

This can be followed by a PCR to assemble the VH and VL sequences into asuitable antibody format for display, such as an scFv. Subsequently, theconstruct can be ligated into a suitable display vector, such as aphagemid vector.

In some embodiments, the nucleic acids of step (a), “providing at leastone nucleic acid encoding a non-human-derived complementaritydetermining region 3 (CDR3) amino acid sequence or an amino acidsequence further encompassing 1, 2, or 3 amino acids N-terminal and/orC-terminal of the non-human-derived CDR3 amino acid sequence,” areprovided by: (1) determining the nucleic acid sequence(s) of at leastone CDR3 region, preferably CDR-3H and CDR-3L region, of at least onenon-human antibody, or of a region further comprising 1, 2, or 3 aminoacids N-terminal of the non-human CDR3 amino acid sequence; (2)providing at least one oligonucleotide, which has the followingstructure: FR3′-CDR3-FR4′, wherein FR3′ represents a sequence encoding ahuman FR3 region, or a fragment thereof comprising the C-terminal end ofthe FR3 region, and wherein FR4′ represents a sequence encoding a humanFR4 region, or a fragment thereof comprising the N-terminal end of theFR4 region; (3) generating the population of nucleic acids of (b) byPCR, in particular encompassing overlap PCR.

Determining the nucleic acid sequence of a CDR3 region of gene encodingan antibody can be performed by sequencing methods known in the art.

An oligonucleotide can be produced by methods known in the art, such assolid phase synthesis.

In a preferred embodiment, the population of nucleic acids encodesproteins comprising at least a VH domain and at least one VL domain,more preferably the nucleic acids encode an scFv.

In a preferred embodiment, step (3) comprises: generating a populationof nucleic acids encoding at least one variable domain by PCR using apopulation of template nucleic acids, wherein the template nucleic acidscomprise Acceptor Framework nucleic acids of the disclosure.

Thereby, a library comprising a VH domain, or a VL domain, respectively,is generated.

In a further preferred, a population of nucleic acids encoding a proteincomprising a VH domain and a VL domain, in particular an scFv, accordingto the disclosure is generated.

Therefore, in a more preferred embodiment, step (3) above furthercomprises generating nucleic acids encoding a protein comprising a VHdomain and a VL domain, in particular an scFv, by overlap PCR.

It is understood that FR3 and FR4 are heavy chain FR3 and FR4 in case ofCDR-3H, and that FR3 and FR4 are light chain FR3 and FR4 in case ofCDR-3L.

In a preferred embodiment, the nucleic acid sequence moieties FR3′ andFR4′, respectively, both independently have a length which allows forstable base pairing with the corresponding complementary strand undersuitable conditions. In particular, the length of FR3′ and FR4′independently is at least about 15 nucleotides, preferably at leastabout 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 43, 35 or more nucleotides. For example, the length is up to50, 75 or 100 nucleotides. For example, FR3′ may be a nucleic acidencoding human FR3. For example, FR4′ may be a nucleic acid encodinghuman FR4.

Therefore, in a preferred embodiment, step (a) of the above methodcomprises: generating a population of nucleic acids comprising: (i) asequence encoding a human FR3 region, or a fragment thereof comprisingthe C-terminal end of the FR3 region; (ii) a sequence encoding anon-human-derived CDR3 amino acid sequence or an amino acid sequencefurther encompassing 1, 2, or 3 amino acids N-terminal and/or C-terminalof the non-human-derived CDR3 amino acid sequence; and (iii) a sequenceencoding a human FR4 region, or a fragment thereof comprising theN-terminal end of the FR4 region, with the proviso that the twoC-terminal amino acids of FR3 are optionally non-human.

Alternatively, Acceptor Framework nucleic acid sequence may be provided,which comprise FR1, FR2, FR3 and FR4 regions, and CDR1 and CDR2 regionsof the disclosure, respectively. Non-human-derived CDR3 regions may becloned into the Acceptor Framework nucleic acids by suitable methods.Therefore, in a further preferred embodiment, step (b) of the abovemethod comprises: (i) providing a population of Acceptor Frameworknucleic acid sequences, wherein each Acceptor Framework nucleic acidsequence comprises nucleic acid sequences encoding a set of frameworkregions comprising a first human framework region (FR1), a second humanframework region (FR2), a third human framework region (FR3), and afourth human framework region (FR4), wherein the FR1 and FR2 regions areinterspaced by a complementarity determining region 1 (CDR1), the FR2and FR3 regions are interspaced by a complementarity determining region2 (CDR2), and the nucleic acid sequences encoding FR3 and FR4 regionsare linked directly or are interspaced by a stuffer nucleic acidsequence, and (ii) combining at least one nucleic acid sequence encodinga non-human-derived CDR3 amino acid sequence with an Acceptor Frameworknucleic acid sequence, so that the FR3 and FR4 regions are interspacedby a non-human-derived CDR3 amino acid sequence or an amino acidsequence further encompassing 1, 2, or 3 amino acids N-terminal and/orC-terminal of the non-human-derived CDR3 amino acid sequence, with theproviso that the two C-terminal amino acids of FR2 are optionallynon-human, and that the two C-terminal amino acids of FR3 are optionallynon-human.

An “Acceptor Framework nucleic acid” according to the present disclosurerefers to a nucleic acid sequence that comprises the nucleic acidsequences encoding the FR1, FR2, FR3 and FR4 regions, and the nucleicacid sequences encoding a CDR1 and a CDR2 region or amino acid sequencesthat can fulfill the role of these CDRs, as defined herein, with thestructure FR1-CDR1-FR2-CDR2-FR3-L-FR4, wherein L is either a directlinkage or a stuffer nucleic acid sequence, which direct linkage orstuffer nucleic acid sequence serves as the site of integration for anucleic acid encoding a non-human-derived CDR3 amino acid sequence,preferably a non-human-CDR3 amino acid sequence, or a non-human-derivedCDR3 amino acid sequence, preferably a non-human-CDR3 amino acidsequence further comprising 1, 2, or 3 amino acids N-terminal of thenon-human-derived CDR3 amino acid sequence, and/or 1, 2, or 3 aminoacids C-terminal of the non-human-derived CDR3 amino acid sequence.

Accordingly, an “Acceptor Framework” according to the present disclosurerefers to a protein comprising the FR1, FR2, FR3 and FR4 regions, andthe CDR1 and CDR2 regions, or amino acid sequences that can fulfill therole of these CDRs, as defined herein, with the structureFR1-CDR1-FR2-CDR2-FR3-L-FR4, wherein L is either a direct peptidelinkage or a stuffer sequence, wherein the corresponding nucleic aciddirect linkage or stuffer nucleic acid sequence serves as the site ofintegration for a nucleic acid encoding a non-human-derived CDR3 aminoacid sequence, preferably a non-human-CDR3 amino acid sequence, or anon-human-derived CDR3 amino acid sequence, preferably a non-human-CDR3amino acid sequence further comprising 1, 2, or 3 amino acids N-terminalof the non-human-derived CDR3 amino acid sequence, and/or 1, 2, or 3amino acids C-terminal of the non-human-derived CDR3 amino acidsequence. The Acceptor Framework can be a variable heavy chain (VH)Acceptor Framework or a variable light chain (VL) Acceptor Framework, orcan be a Framework comprising a variable heavy chain (VH) AcceptorFramework and a variable light chain (VL) Acceptor Framework, such as anscFv Acceptor Framework comprising insertion sites for CDR-3H andCDR-3L.

In a further preferred embodiment, the nucleic acid sequences encodingthe non-human-derived CDR3 amino acid sequences or the amino acidsequence further encompassing 1, 2, or 3 amino acids N-terminal and/orC-terminal of the non-human-derived CDR3 amino acid sequence arediversified among the population of nucleic acids encoding at least oneprotein comprising at least one immunoglobulin variable domain.

“The nucleic acid sequences encoding the non-human-derived CDR3 aminoacid sequences or the amino acid sequence further encompassing 1, 2, or3 amino acids N-terminal and/or C-terminal of the non-human-derived CDR3amino acid sequence are diversified among the population of nucleicacids” is understood as that at least 2 of the nucleic acids of thepopulation encode different CDR3 amino acid sequences, in particulardifferent CDR-3H and/or CDR-3L amino acid sequences, more preferablywherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 or moreencode different CDR3 amino acid sequences, in particular differentCDR-3H and/or CDR-3L amino acid sequence, and/or wherein at least 50%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% of the nucleic acids of the populationencode different CDR3 amino acid sequences, in particular differentCDR-3H and/or CDR-3L amino acid sequences. In a more preferredembodiment the, sequences are diversified due to immunization of one ormore non-humans with a target of interest. Preferably, the sequences arediversified within the CDR3 amino acid sequence, in case the sequencefurther encompasses 1, 2, or 3 amino acids N-terminal and/or C-terminalof the non-human-derived CDR3 amino acid sequence.

It is preferred to use the sequences identical to the CDR3 regions ofnon-human antibodies raised against the antigen of interest. Asdescribed above, the nucleic acids encoding a non-human CDR3 region canbe obtained such as by amplification (e.g. by PCR methods), or bychemical synthesis. Alternatively, an SDR region may be cloned. Such SDRregion encompasses 3 further amino acids N-terminal and 1 amino acidC-terminal of CDR3 in case of CDR-3H, as shown above. By using thenon-human CDR3 regions or a sequence further encompassing 1, 2, or 3amino acids N-terminal and 1 amino acid C-terminal of the non-human CDR3amino acid sequence, efficient mass humanization of the non-humanantibodies can be achieved, with a high likelihood of obtaining asuccessful antibody graft for the CDR3. An SDR is known as a CDR plus anadditional few additional boundary residues known in art as “vernierzones”.

In a further preferred embodiment, a nucleic acid sequence encoding anon-human-derived CDR3 amino acid sequence is a nucleic acid sequenceencoding a non-human CDR3 amino acid sequence, or an amino acid sequencefurther encompassing 1, 2, or 3 amino acids N-terminal and 1 amino acidC-terminal of the non-human CDR3 amino acid sequence.

In a preferred embodiment, the CDR3 amino acid sequence is a non-humanCDR3 amino acid sequence. Such non-human sequences are naturallyoccurring in the non-human. Antibodies are preferably antibodiesproduced by B cells, in particular after immunization of the non-humanwith an antigen of interest. Suitable immunization protocols andprotocols for isolating sources of B cells are known in the art, such asbone marrow cells, PBMC cells or spleen cells. Therefore, in an evenmore preferred embodiment, the non-human CDR3 amino acid sequence, or anamino acid sequence further encompassing 1, 2, or 3 amino acidsN-terminal and 1 amino acid C-terminal of the non-human CDR3 amino acidsequence is obtained from a non-human bone marrow cell, PBMC cell orspleen cell, more preferably wherein the non-human was immunized againstan antigen of interest.

In a preferred embodiment, it is possible to use a plurality or all ofthe non-human CDR3 sequences determined in one or more non-humansimmunized with an antigen of interest.

In further preferred embodiments, a preselection of non-human CDR3sequences is performed in step (a). Such preselection can be performedas follows: (i) RNA or DNA is extracted from a B cell source of at leastone non-human immunized against an antigen of interest, such asnon-human spleen, bone marrow, blood, or the lymph node; (ii) nucleicacids encoding the protein sequence comprising the CDR-3H and optionallyCDR-3L regions of the antibody repertoire of the non-humans areamplified; in particular the VH and VL Fav fragment, or minimally theCDR-3H and optionally CDR-3L sequences are amplified; (iii) theamplified products are sequenced; (iv) the resulting sequences areanalyzed to identify the translated or untranslated CDR-3H andoptionally CDR-3L sequences; (v) the frequency of the CDR-3H andoptionally CDR-3L sequences, respectively, of the analyzed repertoire isanalyzed, and trees of related CDR-3H and optionally CDR-3L sequences,respectively, are generated by single linkage; (vi) optionally, CDR-3Hand optionally CDR-3L sequences which are also determined in a sampleobtained from the at least one non-human prior to immunization areexcluded; (vii) candidate lineages are ranked by expansion, isotope,somatic hypermutation, tree complexity, and convergence; (viii)individual representatives of each lineage are selected and synthesized,wherein silent or non-silent mutations or natural degeneracy can beincorporated during synthesis; (viii) generating a nucleic acidpopulation of the disclosure comprising the non-human CDR3 regions, e.g.by methods described above.

Therefore, in a particularly preferred embodiment, the non-human CDR3amino acid sequence, or an amino acid sequence further encompassing 1,2, or 3 amino acids N-terminal and 1 amino acid C-terminal of thenon-human CDR3 amino acid sequence is obtained by: (i) determining thesequence of the non-human CDR3 regions of the antibodies in a sampleobtained from a non-human immunized against an antigen of interest; (ii)determining the frequency of all non-human CDR3 amino acid sequence inthe sample and generating lineage trees; (iii) optionally excludingnon-human CDR3 amino acid sequence sequences present in a sample fromthe non-human prior to immunization; (iv) ranking candidate lineages byexpansion, isotype, somatic hypermutation, tree complexity, and/orconvergence; (v) selecting an individual non-human CDR3 amino acidsequence representative of at least one lineage, in particular of aplurality of lineages or all lineages; and (vi) generating a nucleicacid encoding a peptide comprising the individual non-human CDR3 aminoacid sequence, or a derivative thereof containing 1, 2 or 3 conservativeamino acid mutations.

As described above, it is possible that the two C-terminal amino acidsof the FR3, in particular of heavy FR3 are non-human, in particular incase an SDR encompassing a non-human CDR-3H is amplified and grafted toobtain the nucleic acids of the population of the disclosure. As alsodescribed above, it is possible that two C-terminal amino acids of theFR2 region are non-human.

It is, however, also possible to incorporate sequences encoding peptideconsisting of a non-human-derived complementarity determining region 3(CDR3) amino acid sequence. In this embodiment, it is preferred that theFR3 region, in particular the heavy FR3 region and the light FR3 regionis human. Further, it is possible to use a human FR2 region. Therefore,in another preferred embodiment, the FR2 region is human, and/or the FR3region is human.

The methods herein encompass in one preferred embodiment the followingsteps:

Step 1: Cloning of Non-human VH-CDR3 regions between Human VH-FR3 andHuman VH-FR4 regions in an acceptor vector

Step 2: Transformation of E. coli with the ligation from step 1 togenerate a Non-human VH-CDR3 library between Human VH-FR3 and HumanVH-FR4 regions

Step 3: PCR amplification using DNA template from transformed bacteriafrom step 2 of Non-human VH-CDR3 library between Human VH-FR3 and HumanVH-FR4 regions

Step 4: Assembly of Non-human VL-CDR3 regions into an acceptor vectorcontaining synthesized human FR1, FR2 and FR3 domains and a library ofCDR1 and CDR2 sequences

Step 5: Transformation of E. coli with the ligation from step 4 togenerate a Non-human VL-CDR3 library between Human VL-FR3 and HumanVL-FR4 regions

Step 6: PCR amplification using a synthesized DNA template containingHuman FR1, FR2 and FR3 domains and a library of VH-CDR1 and VH-CDR2

Step 7: Assembly of a VH variable region library containing HumanFramework regions FR1, FR2 and FR3 separated by a library of CDR1 andCDR2 sequences and a library of Non-human CDR3 sequences via overlap PCR

Step 8: PCR of the VH variable region library from step 7 containingHuman Framework regions FR1, FR2, FR3 and FR4 separated by a library ofCDR1, CDR2 and a library of Non-human CDR3 sequences

Step 9: PCR amplification of a VL variable region library containing theC-terminal part of a Human VH-FR4 domain, a linker sequence, Human VLFramework domain regions FR1, FR2, FR3 and FR4 separated by a library ofCDR1 and CDR2 sequences and a library of Non-human VL-CDR3

Step 10: PCR assembly via overlap PCR of DNA fragments derived fromsteps 8 and 9 via their common human VH-FR4 sequence

Therefore, a novel mass humanized library of scFv fragments comprisingnon-human CDR3 regions is generated.

Optionally, the library may be cloned into a suitable display vector ina subsequent step.

Step 11: Cloning of the assembled scFv library in a phage displayvector, or into another suitable display vector, such as a vector forribosome display, or yeast display.

In a yet further preferred embodiment, the diversified non-human-derivedCDR3 amino acid sequences or the amino acid sequence furtherencompassing 1, 2, or 3 amino acids N-terminal and/or C-terminal of thenon-human-derived CDR3 amino acid sequence encode light chain CDR3 (CDRL3) sequences. In a particularly preferred embodiment, the light chainCDR3 (CDR L3) sequences have a length of between 5 to 20 amino acids,even more preferably between 7 and 13 amino acids.

In some embodiments, the nucleic acids encoding the CDR1 and CDR2 aminoacid sequences are diversified among the population of nucleic acidsencoding at least one protein comprising at least one immunoglobulinvariable domain, wherein each nucleic acid sequence encoding a CDR1 orCDR2 amino acid sequence is independently based i) on a nucleic acidsequence encoding a human CDR1 or CDR2, respectively, or ii) on anucleic acid sequence encoding a non-human CDR1 or CDR2, respectively,wherein at least some of the nucleic acid sequences encoding a CDR1 orCDR2 amino acid sequence have been modified to encode at least one aminoacid present in non-human CDR1 or CDR2 amino acid sequences,respectively, in case of human CDR1 or CDR2, respectively, or to encodeat least one amino acid present in human CDR1 or CDR2 amino acidsequences, respectively, in case of non-human CDR1 or CDR2,respectively.

A suitable method for obtaining diversified CDR1 and CDR2 sequencesaccording to the disclosure encompass computationally integrating aplurality, such as 10⁴, 10⁵, or 10⁶-10⁷ of simulated humanizationsacross the non-human antibody repertoire and the human acceptor antibodyrepertoire, in order to generate a mathematically optimal Bayesianrepresentation of the humanization space between non-human and human,converting the probabilistic model to a frequentist interpretation,thereby obtaining a population or library. Each position ends upcontaining a probability of encountering the human germline residue, thenon-human germline residues, and the most common collection of affinitymaturation residues from both species. A map can be generated betweenevery observed antibody in the non-human species and every preferredacceptor scaffold in humans. All sequences from both species can then beanalyzed to: (1) obtain an optionally non-redundant database of aminoacid sequences from both species from at least the memory B cellrepertoire; (2) identify preferred human acceptor scaffolds for eachnon-human variable gene; (3) generate a positional weight matrix (PWM)of amino acid positional variability in the CDR1 and CDR2 of thenon-human species and the human species by calculating the relativefrequency of each amino acid at each position of a specific non-humanvariable gene and each member of the subset of preferred acceptorscaffolds from humans (see, e.g. FIG. 3); (4) blend each of two PWMs(from one or more amino acids) from the non-human variable gene and avariable gene from the human preferred acceptor scaffolds to produce ahybrid PWM that contains the amino acid variation observed from bothspecies at each position; (4) optionally adjust the blended PWM toremove cysteine, methionine, and tryptophan residues in order togenerate superior therapeutics; and (5) optionally adjust thecomposition of the blended PWM to bias the library more towards eitherthe human or non-human molecules, as well as to adjust the effectivediversity of the library. In some embodiments, amino acid contributionsfrom the human and non-human species are weighted evenly. In someembodiments the frequencies are altered such that in any combination ofa set of amino acids from a collection of CDR sequences, a set ofnon-human example antibodies would occur at a frequency greater than theinverse of the size of the library or a predetermined set threshold. Theset of amino acids from a collection of CDR sequences can be 5 aminoacids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 aminoacids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids,19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 aminoacids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids,28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 aminoacids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids,37 amino acids, 38 amino acids, 39 amino acids, or 40 amino acids. Insome embodiments the predetermined set threshold can be 10e-6, 10e-7,10e-8, or 10e-9. In some embodiments, the human or non-human amino acidcontributions are weighted so as to increase their representationrelative to the other, such as by about or more than about 0.1%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more. The PWMs can beconverted to instructions to produce a library of nucleic acid moleculesencoding immunoglobulin molecules. Nucleic acid synthesis instructionsare generated such that each amino acid is encoded at a position atapproximately its frequency within the PWM, with the frequency of eachamino acid position independent of the others. The in silico nucleicacid library produced by these methods can be analyzed by probabilisticsimulation to analyze the predicted properties of the synthesizedlibrary. The expected frequency of every molecule in the library can becalculated. Mathematical simulation can be used to explore thetheoretical humanization of non-human antibodies to evaluate theproximity of the closest humanizations, and the blended PWM can beadjusted to bias the libraries towards a given level of humanization.This can be accomplished by iterative Monte Carlo sampling or othermethods.

In a preferred embodiment, cysteine, and/or methionine residues areremoved and/or the asparagine residue content is reduced, as theseresidues form biochemical liabilities.

By analyzing the CDR1 and CDR2 repertoires of non-human and human,respectively, it was possible to identify and generate CDR1 and CDR2diversified sequences that span non-human germline, human germline,non-human somatic hypermutation, human somatic hypermutation, andnon-human gene conversion at every position in CDR-H1, CDR-H2, CDR-L1,and CDR-L2. The resulting population or library thus is a novel entitywith surprisingly beneficial properties, that is neither non-human norhuman, but a hybrid repertoire exploring the space between.

Therefore, in a yet further preferred embodiment, the human or non-humanCDR1 regions and the human and non-human CDR2 regions, on which thediversified CDR1 and CDR2 amino acid sequences are based, are selectedfrom human germline CDR1 regions, human germline CDR2 regions, non-humangermline CDR1 regions, non-human germline CDR2 regions, human somatichypermutation CDR1 regions, human somatic hypermutation CDR2 regions,non-human somatic hypermutation CDR1 regions, non-human somatichypermutation CDR2 regions, non-human gene conversion CDR1 regions, andnon-human gene conversion CDR2 regions.

In a yet further preferred embodiment, the diversified CDR1 and CDR2amino acid sequences are CDR-H1, CDR-H2, CDR-L1 and/or CDR-L2 sequences.

In a preferred embodiment, the population includes at least one nucleicacid encoding a human CDR-H1, and/or a human CDR-H2, and/or a humanCDR-L1 and/or a human CDR-L2 sequence.

In a further preferred embodiment, the population includes at least onenucleic acid encoding a non-human CDR-H1, and/or a non-human CDR-H2,and/or a non-human CDR-L1 and/or a non-human CDR-L2 sequence.

In some embodiments, the human FR1, FR2, FR3 and FR4 regions are humanframework regions selected to provide a scaffold conducive for non-humanCDR3 amino acid sequences, with the proviso that the two C-terminalamino acids of FR2 are optionally non-human, and that the two C-terminalamino acids of FR3 are optionally non-human.

Therefore, in one preferred embodiment, the population comprises atleast one nucleic acid encoding a human FR1, a human FR2, a human FR3and a human FR4 region, and a human CDR1, and a human CDR2 region.

In another preferred embodiment, the population comprises at least onenucleic acid encoding a human FR1, a human FR2, a human FR3 and a humanFR4 region, and a non-human CDR1, and a non-human CDR2 region.

Therefore, in yet another preferred embodiment, the population does notcontain a nucleic acid encoding a human FR1, a human FR2, a human FR3and a human FR4 region, and a human CDR1, and a human CDR2 region.

Therefore, in yet another preferred embodiment, the population does notcontain a nucleic acid encoding a human FR1, a human FR2, a human FR3and a human FR4 region, and a non-human CDR1, and a non-human CDR2region.

A suitable scaffold conducive for non-human CDR3 amino acid sequencesmay be obtained by selecting a framework set by performing the followingsteps:

computational imputation of germline repertoire element centroids whichare most utilized in functional non-human antibodies

generating an amino acid alignment of human frameworks compared to saidcomputationally imputed non-human frameworks and selecting the closestrepresentatives, respectively, that have the same length of CDR-H1,CDR-H2, CDR-L1, and CDR-L2 and similar sequence composition,respectively,

further reducing the number of candidates framework sequences bypreferring templates that are known to be stable in the art andoptionally have worked as previous drugs,

further reducing the number of framework candidates by structurallymodeling antibodies from non-human and human using these frameworks andanalyzing their structural superposition tolerance by root mean squareddeviation (RMSD), and

selecting a set of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heavy chain and 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 light chain frameworks comprising set of FR1,FR2, FR3 and FR4 regions, respectively.

As an example, a set of 2 heavy chain and 2 light chain frameworkscomprising set of FR1, FR2, FR3 and FR4 regions respectively, can beselected. Moreover, the two C-terminal amino acids of heavy chain FR2are optionally non-human, and the two C-terminal amino acids of heavychain FR3 are optionally non-human, for example in case an SDR isgrafted into a scaffold, or in case amino acids surrounding FR2 aresubstituted, as described above.

Therefore, in a yet further preferred embodiment, the human FR1, FR2,FR3 and FR4 regions which are human framework regions selected toprovide a scaffold conducive for non-human CDR3 amino acid sequences areobtainable by:

(i) providing

(a) a collection of sequences of naturally occurring human antibodieseach comprising a set of human FR1, FR2, FR3 and FR4 regions; and

(b) a collection of sequences of naturally occurring non-humanantibodies each comprising a set of non-human FR1, FR2, FR3 and FR4regions, and

(ii) identifying a plurality of sets of human FR1, FR2, FR3 and FR4regions which provide a scaffold conducive for non-human CDR3 amino acidsequences by

determining the parameters framework homology, CDR cosmology, CDRlengths, CDR canonical structure, and adoption of comfortable heavy orlight mount angles, and

selecting sets of human FR1, FR2, FR3 and FR4 regions which exhibit highscores for the parameters,

and/or

the two C-terminal amino acids of heavy chain FR2 are optionallynon-human, and

the two C-terminal amino acids of heavy chain FR3 are optionallynon-human.

In a further embodiment, the present disclosure provides a population ofnucleic acids encoding at least one protein comprising at least oneimmunoglobulin variable domain having a non-human-derived CDR3 aminoacid sequence embedded in essentially human framework sequences,obtainable by a method described herein.

Such population preferably represents a library of non-human CDR3sequences, wherein mass humanization of the non-human antibodies isachieved.

It is understood that the preferred embodiments described for themethods of the disclosure also apply for the populations obtainedthereby, and uses thereof.

In a preferred embodiment, a nucleic acid of the population is locatedin a vector. Such vector allows easy and efficient replication, cloning,selection and/or display, depending on the properties of such vector.Accordingly, a vector may additionally include nucleic acid sequencesthat permit it to replicate in the host cell, such as an origin ofreplication, one or more therapeutic genes and/or selectable markergenes and other genetic elements known in the art such as regulatoryelements directing transcription, translation and/or secretion of theencoded protein. The vector may be used to transduce, transform orinfect a cell, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell. The vector optionallyincludes materials to aid in achieving entry of the nucleic acid intothe cell, such as a viral particle, liposome, protein coating or thelike. Numerous types of appropriate expression vectors are known in theart for protein expression, by standard molecular biology techniques.Such vectors are selected from among conventional vector types includinginsects, e.g., baculovirus expression, or yeast, fungal, bacterial orviral expression systems. Other appropriate expression vectors, of whichnumerous types are known in the art, can also be used for this purpose.Methods for obtaining such expression vectors are well-known (see, e.g.Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition, ColdSpring Harbor Laboratory, New York (1989)). In one embodiment, thevector is a viral vector. Viral vectors include, but are not limited to,retroviral and adenoviral vectors.

Suitable host cells or cell lines for transfection include bacterialcells. For example, the various strains of E. coli are well-known ashost cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas, Streptomyces, and other bacilli and the like mayalso be employed. Many strains of yeast cells known to those skilled inthe art are also available as host cells for expression of peptidesdisclosed herein. Other fungal cells or insect cells such as Spodopterafrugipedera (Sf9) cells may also be employed as expression systems.Alternatively, mammalian cells, such as human 293 cells, Chinese hamsterovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cellsderived from Swiss, BALB/c or NIH mice may be used. Still other suitablehost cells, as well as methods for transfection, culture, amplification,screening, production, and purification are known in the art.

The host cells can be transfected, e.g. by conventional means such aselectroporation with at least one expression vector containing a nucleicacid of the population of the disclosure under the control of atranscriptional regulatory sequence. The transfected or transformed hostcell is then cultured under conditions that allow expression of theprotein. The expressed protein(s) is(are) recovered, isolated, andoptionally purified from the cell or from the culture medium, ifexpressed extracellularly by appropriate means known to one of skill inthe art. For example, the proteins are isolated in soluble formfollowing cell lysis, or extracted using known techniques, e.g. inguanidine chloride. If desired, the proteins are produced as a fusionprotein, in particular in case display of the proteins is intended. Theproteins may be further purified using any of a variety of conventionalmethods including, but not limited to: liquid chromatography such asnormal or reversed phase, using HPLC, FPLC and the like; affinitychromatography such as with inorganic ligands or monoclonal antibodies;size exclusion chromatography; immobilized metal chelate chromatography;gel electrophoresis; and the like. One of skill in the art may selectthe most appropriate isolation and purification techniques.

In a more preferred embodiment, the nucleic acids are comprised in anexpression vector suitable for display of the protein encoded by thenucleic acid on a virus, a cell or a surface. Typically, the nucleicacids encode fusion proteins comprising a protein comprising at leastone immunoglobulin variable domain having a non-human-derived CDR3 aminoacid sequence embedded in essentially human framework sequences and aprotein sequence which allows display on a virus, a cell or a surface.

In a further embodiment, the present disclosure proves a population ofproteins comprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences, obtainable by

(i) expressing at least one protein encoded by the population describedabove in a suitable expression system, and

(ii) optionally displaying at least one protein on a virus, a cell or asurface.

The cell is preferably a bacterial cell or a eukaryotic cell, such as ayeast cell.

The preferred embodiments for methods of the disclosure also apply topopulations of proteins described herein.

In a further embodiment, the present disclosure provides a population ofproteins comprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences, obtainable by

(i) expressing at least one protein encoded by a population as describedabove in a suitable expression system, and

(ii) displaying at least one protein on a virus, a cell or a surface.

Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 ormore proteins encoded by a population of the present disclosure areexpressed in a suitable expression system.

In a more preferred embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50,100, 150, 200 or more proteins encoded by a population of the disclosureare displayed on a virus, a cell or a surface, preferably wherein theproteins are fusion proteins, such as a fusion protein to a minor coatprotein of a bacterial phage or to Agap2p.

A number of display techniques are known in the art, which enable aconnection between genotype and binding properties of the antibodies.For example, display may be achieved by phage display, yeast display,bacterial display, ribosome display mRNA. In some embodiments, thedisplay technique is phage display. In a typical phage display, theprotein comprising the antigen of interest is expressed as a polypeptidefusion to a bacteriophage coat protein and subsequently screened bybinding to immobilized or soluble biotinylated ligand (e.g., Huse etal., '89; Clackson et al., '91; Marks et al., '92). Fusions are mademost commonly to a minor coat protein, called the gene III protein(pIII), which is present in three to five copies at the tip of thephage. A phage constructed in this way can be considered a compactgenetic “unit,” possessing both the phenotype (binding activity of thedisplayed antibody) and genotype (the gene coding for that antibody) inone package. Phage display has been successfully applied to antibodies,DNA binding proteins, protease inhibitors, short peptides, and enzymes.

Antibodies possessing desirable binding properties are preferablyselected by binding to immobilized antigen in a process called“panning”. Phage-bearing nonspecific antibodies are removed by washing,and then the bound phage are eluted and amplified by infection of E.coli. This approach has been applied to generate antibodies against manyantigens.

Yeast display methods are well-known to a skilled person and are forexample described in WO 99/36569. Typically, fusion proteins comprisingthe yeast protein Aga2p are used for displaying the proteins of interestat the cell surface.

Ribosome display techniques are also known in the art and are forexample described in Hanes, J.; Plückthun, A. (1997; Proc. Natl. Acad.Sci. U.S.A. 94 (10): 4937-42) and He M. and Taussig M. J. (2007; NatureMethods 4 (3): 281-288).

In a further embodiment, the present disclosure provides a librarycomprising a plurality, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100,150, 200, 10³, 10⁴, 10⁵, 10⁶ or more of replicable genetic packages,that are capable of displaying at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 150, 200 or more proteins encoded by a population of thedisclosure on a virus, a cell or a surface.

In a further embodiment, the present disclosure provides a librarycomprising a plurality, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100,150, 200, 10³, 10⁴, 10⁵, 10⁶ or more of replicable genetic packages,that display at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200or more proteins encoded by a population of the disclosure on a virus, acell or a surface.

A replicable genetic package is understood as a biological complexcomprising a nucleic acid, and at least one peptide encoded by thenucleic acid. Examples of replicable genetic packages include cells,spores, bacteria, viruses and bacteriophage. Thus, the particularreplicable genetic package or library thereof can be selected from anyone of the foregoing and/or include different combinations thereof.Replicable genetic packages are capable of replication either byself-replication, in combination with a host and/or a helper virus, orby in vitro replication, transcription and expression.

Bacteriophages including phagemids are preferred replicable geneticpackages. Preferred phage are the filamentous phage (e.g., M13, fd andfl) and phagemid vectors derived therefrom. See, WO 91/19818; WO91/18989; WO 92/01047; WO 92/06204; WO 92/18619. Other phage of E. coli,such as T7 phage, or phage of other bacterial species can also be used.Filamentous phages are 6 nm in diameter and up to one micron in length.It has been used extensively in peptide phage display. Its surfaceconsists of five coat proteins, two of which, pIII and pVIII, have beenused to display peptide libraries, pIII contains 406 amino acids and ispresent in three to five copies. The major coat protein, pVIII, whichcontains 50 amino acids, constitutes the bulk of the phage protein as itis present in approximately 2700 copies. The bacteriophage can also be anon-filamentous phage such as icosahedral phages T7 and lambda. Themajor coat protein of T7 phage is the gene 10 capsid protein, whichcontains 370 amino acids and is present in 415 copies.

In addition to phage, a replicable genetic package of the disclosure caninclude eukaryotic viruses (e.g. the Moloney murine leukemia virus; see,e.g., Han, et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995)) orspores (e.g. spores from B. subtilis; see, e.g., Donovan, et al., J.Mol. Biol. 196:1-10 (1987)). A variety of different cells can also beused as replicable genetic packages. Examples of suitable bacterialcells include, but are not limited to, Salmonella typhimurium, Bacillussubtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia,Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus,Moraxella bovis, and especially Escherichia coli.

In a yet further embodiment, the present disclosure provides a methodfor screening for at least one protein comprising at least oneimmunoglobulin variable domain, in particular antibodies or fragmentsthereof, in particular selected from Fab, scFv and Fv, whichspecifically binds to an antigen of interest, comprising the followingsteps:

a) providing a library comprising a plurality, preferably 2, 3, 4, 5, 6,7, 8, 9, 10, 50, 100, 150, 200, 10³, 10⁴, 10⁵, 10⁶ or more of replicablegenetic packages that display at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 150, 200 or more proteins encoded by a population of thedisclosure on a virus, a cell or a surface,

b) contacting the library of a) with at least one antigen of interest ora fragment thereof comprising at least one potential epitope,

c) isolating at least one genetic package which exhibits the desiredbinding property, in particular affinity, and

d) optionally determining the sequence or part of the sequence of thenucleic acid encoding the protein comprising at least one immunoglobulinvariable domain,

e) optionally repeating steps a) to d) one or more times with 2 or moregenetic packages isolated in step c).

As explained above, affinity to the antigen may be determined. Forexample, this can be performed by surface plasmon resonancespectroscopy, e.g. using a Biacore apparatus. For example, proteinsexhibiting an affinity (Kd) of 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less may beselected and optionally used in further screening rounds.

In a yet further embodiment, a method of the present disclosure may berepeated one or more times, for example 1, 2, 3, 4, 5 or more times.Thereby, additional selection rounds are performed.

In a preferred embodiment, at least 2 of the displayed proteins havedifferent CDR3 amino acid sequences, in particular different CDR-3Hand/or CDR-3L amino acid sequences,

more preferably wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100,150, 200 or more of the displayed proteins have different CDR3 aminoacid sequences, in particular different CDR-3H and/or CDR-3L amino acidsequences, and/or

wherein at least 50%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% of the nucleicacids of the of the displayed proteins have different CDR3 amino acidsequences, in particular different CDR-3H and/or CDR-3L amino acidsequences.

In a further embodiment, the present disclosure provides a population ofnucleic acids encoding at least one protein comprising at least oneimmunoglobulin variable domain having a non-human-derived CDR3 aminoacid sequence embedded in essentially human framework sequences, whereinthe human framework sequences comprise a first human framework region(FR1), a second human framework region (FR2), a third human frameworkregion (FR3), and a fourth human framework region (FR4), such that theFR1 and FR2 regions are interspaced by a complementarity determiningregion 1 (CDR1), the FR2 and FR3 regions are interspaced by acomplementarity determining region 2 (CDR2), and the FR3 and FR4 regionsare interspaced by a non-human-derived CDR3 amino acid sequence; whereinthe nucleic acid sequences encoding the CDR1 and CDR2 amino acidsequences are diversified among the population of nucleic acids encodingat least one protein comprising at least one immunoglobulin variabledomain, wherein each nucleic acid sequence encoding a CDR1 or CDR2 aminoacid sequence is independently based (i) on a nucleic acid sequenceencoding a human CDR1 or CDR2, respectively, or (ii) on a nucleic acidsequence encoding a non-human CDR1 or CDR2, respectively; wherein atleast some of the nucleic acid sequences encoding a CDR1 or CDR2 aminoacid sequence have been modified to encode at least one amino acidpresent in non-human CDR1 or CDR2 amino acid sequences, respectively, incase of human CDR1 or CDR2, respectively, or to encode at least oneamino acid present in human CDR1 or CDR2 amino acid sequences,respectively, in case of non-human CDR1 or CDR2, respectively; andwherein the human FR1, FR2, FR3 and FR4 regions are human frameworkregions selected to provide a scaffold conducive for non-human CDR3amino acid sequences, with the proviso: that the two C-terminal aminoacids of FR2 are optionally non-human, and that the two C-terminal aminoacids of FR3 are optionally non-human.

It is understood that the preferred embodiments for the methods of thedisclosure also apply to the populations of nucleic acids describedherein.

In particular, the human FR1, FR2, FR3 and FR4 regions are humanframework regions with the proviso that the two C-terminal amino acidsof FR2 are optionally non-human, and that the two C-terminal amino acidsof FR3 are optionally non-human.

Human FR1, FR2, FR3 and FR4 regions are FR1, FR2, FR3 and FR4 regionswhich are naturally occurring in humans. As described above, the twoC-terminal amino acids of FR2 are optionally non-human, and that the twoC-terminal amino acids of FR3 are optionally non-human. In someembodiments, the FR1 and FR4 regions are human FR1 and FR4 regions. Asalso described above, it is possible that a nucleic acid encoding anon-human-derived CDR3 amino acid sequence, preferably a non-human-CDR3amino acid sequence, further comprising 1, 2, or 3 amino acidsN-terminal of the non-human-derived CDR3 amino acid sequence, and/or 1,2, or 3 amino acids C-terminal of the non-human-derived CDR3 amino acidsequence is embedded, in particular in case a nucleic acid encoding anon-human SDR is embedded. For example, an SDR comprising the CDR-3H maybe embedded.

The N-terminal “C” is also present in human heavy FR3 sequences, and theC-terminal “W” is also present in human heavy FR4 sequences.Accordingly, in case the FR3 and FR4 regions are interspaced by anon-human-derived CDR3 amino acid sequence of the structureCAR|XXXXXXXXXXDY| W (SEQ ID NO:1), the resulting FR3 heavy region willcontain the C-terminal non-human amino acids “AR”, whereas the remainingparts of the FR3 heavy region will be human, and the FR4 heavy regionwill be human.

Therefore, in another preferred embodiment, at least one nucleic acidencoding a non-human CDR3 sequence is embedded, such that the human FR3and FR4 regions are interspaced by a non-human CDR3 amino acid sequence.Preferably, also the resulting FR3 and FR4 regions will be human.

Further, FR2 is a human FR2 framework region, or the two C-terminalamino acids of FR2 are optionally non-human. In one preferredembodiment, FR2 is a human FR2 framework region.

In another preferred embodiment, the two C-terminal amino acids of FR2are non-human, whereas the remaining sequence of FR2 is human.

In particular, the two positions N-terminal to CDR-H2 (i.e. positionsX10 and X11) may be “VS” in the human: CDR-H2: GLEWVS|X12X13X14X15X16X17X18X19X20X21X22X23DSVKG|RFT (SEQ ID NO: 2).

In one preferred embodiment, V and/or S at positions X10 and X11 areindependently non-human, preferably non-human, in the heavy FR2 region.

In a further preferred embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 150, 200 or more nucleic acids of the population exhibitdifferent amino acids at the two C-terminal amino acids of human FR2,more preferably at positions X10 and X11 of heavy human FR2.

In a further preferred embodiment, the population of nucleic acidsencodes proteins comprising at least a VH domain and/or at least one VLdomain, more preferably the nucleic acids encode an antibody, an scFv, aFv or Fab.

The nucleic acids encoding the CDR1 and CDR2 amino acid sequences arediversified among the population of nucleic acids encoding at least oneprotein comprising at least one immunoglobulin variable domain, whereineach nucleic acid sequence encoding a CDR1 or CDR2 amino acid sequenceis independently based i) on a nucleic acid sequence encoding a humanCDR1 or CDR2, respectively, or ii) on a nucleic acid sequence encoding anon-human CDR1 or CDR2, respectively, wherein at least some of thenucleic acid sequences encoding a CDR1 or CDR2 amino acid sequence havebeen modified to encode at least one amino acid present in non-humanCDR1 or CDR2 amino acid sequences, respectively, in case of human CDR1or CDR2, respectively, or to encode at least one amino acid present inhuman CDR1 or CDR2 amino acid sequences, respectively, in case ofnon-human CDR1 or CDR2, respectively.

A diversified CDR1 and CDR2 sequences can be obtained by computationallyintegrating a plurality, such as 10⁴, 10⁵, or 10⁶-10⁷ of simulatedhumanizations across the non-human antibody repertoire and the humanacceptor antibody repertoire, in order to generate a mathematicallyoptimal Bayesian representation of the humanization space betweennon-human and human, converting the probabilistic model to a frequentistinterpretation, thereby obtaining a population or library. Each positionends up containing a probability of encountering the human germlineresidue, the non-human germline residues, and the most common collectionof affinity maturation residues from both species.

In a preferred embodiment, cysteine, and/or methionine residues areremoved and/or the asparagine residue content is reduced, as theseresidues form biochemical liabilities.

In a yet further preferred embodiment, the human or non-human CDR1regions and the human and non-human CDR2 regions, on which thediversified CDR1 and CDR2 amino acid sequences are based, are selectedfrom human germline CDR1 regions, human germline CDR2 regions, non-humangermline CDR1 regions, non-human germline CDR2 regions, human somatichypermutation CDR1 regions, human somatic hypermutation CDR2 regions,non-human somatic hypermutation CDR1 regions, non-human somatichypermutation CDR2 regions, non-human gene conversion CDR1 regions, andnon-human gene conversion CDR2 regions.

In a yet further preferred embodiment, the diversified CDR1 and CDR2amino acid sequences are CDR-H1, CDR-H2, CDR-L1 and/or CDR-L2 sequences.

In a preferred embodiment, the population includes at least one nucleicacid encoding a human CDR-H1, and/or a human CDR-H2, and/or a humanCDR-L1 and/or a human CDR-L2 sequence.

In a further preferred embodiment, the population includes at least onenucleic acid encoding a non-human CDR-H1, and/or a non-human CDR-H2,and/or a non-human CDR-L1 and/or a non-human CDR-L2 sequence.

In some embodiments, the human FR1, FR2, FR3 and FR4 regions are humanframework regions selected to provide a scaffold conducive for non-humanCDR3 amino acid sequences, with the proviso that the two C-terminalamino acids of FR2 are optionally non-human, and that the two C-terminalamino acids of FR3 are optionally non-human. In one preferredembodiment, a population of the disclosure comprises at least onenucleic acid encoding a human FR1, a human FR2, a human FR3 and a humanFR4 region, and a human CDR1, and a human CDR2 region.

In another preferred embodiment, a population of the disclosurecomprises at least one nucleic acid encoding a human FR1, a human FR2, ahuman FR3 and a human FR4 region, and a non-human CDR1, and a non-humanCDR2 region.

In yet another preferred embodiment, a population of the disclosure doesnot contain a nucleic acid encoding a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a human CDR1, and a human CDR2 region.

In yet another preferred embodiment, a population of the disclosure doesnot contain a nucleic acid encoding a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a non-human CDR1, and a non-human CDR2region.

A suitable scaffold conducive for non-human CDR3 amino acid sequences isobtainable by selecting a framework set by performing the followingsteps:

computational imputation of germline repertoire element centroids whichare most utilized in functional non-human antibodies

generating an amino acid alignment of human frameworks compared to saidcomputationally imputed non-human frameworks and selecting the closestrepresentatives, respectively, that have the same length of CDR-H1,CDR-H2, CDR-L1, and CDR-L2 and similar sequence composition,respectively,

further reducing the number of candidates framework sequences bypreferring templates that are known to be stable in the art andoptionally have worked as previous drugs,

further reducing the number of framework candidates by structurallymodeling antibodies from non-human and human using these frameworks andanalyzing their structural superposition tolerance by root mean squareddeviation (RMSD), and

selecting a set of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heavy chain and 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 light chain frameworks comprising set of FR1,FR2, FR3 and FR4 regions, respectively.

As an example, a set of 2 heavy chain and 2 light chain frameworkscomprising set of FR1, FR2, FR3 and FR4 regions respectively, can bepresent. Moreover, the two C-terminal amino acids of heavy chain FR2 mayoptionally be non-human, and the two C-terminal amino acids of heavychain FR3 may optionally be non-human, for example in case a specificitydetermining residue (SDR) is grafted into a scaffold, or in case aminoacids surrounding FR2 are substituted, as described above.

In a yet further preferred embodiment, the human FR1, FR2, FR3 and FR4regions which are human framework regions selected to provide a scaffoldconducive for non-human CDR3 amino acid sequences are obtainable by:

(i) providing

(a) a collection of sequences of naturally occurring human antibodieseach comprising a set of human FR1, FR2, FR3 and FR4 regions; and

(b) a collection of sequences of naturally occurring non-humanantibodies each comprising a set of non-human FR1, FR2, FR3 and FR4regions, and

(ii) identifying a plurality of sets of human FR1, FR2, FR3 and FR4regions which provide a scaffold conducive for non-human CDR3 amino acidsequences by

determining the parameters framework homology, CDR cosmology, CDRlengths, CDR canonical structure, and adoption of comfortable heavy orlight mount angles, and

-   -   selecting sets of human FR1, FR2, FR3 and FR4 regions which        exhibit high scores for the parameters,

and/or

the two C-terminal amino acids of heavy chain FR2 are optionallynon-human, and

the two C-terminal amino acids of heavy chain FR3 are optionallynon-human.

In a further embodiment, the present disclosure provides a population ofproteins comprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences, and wherein the proteins further comprise at leastone moiety allowing display of the at least one protein on a virus, acell, or a surface.

Suitable moieties, in particular protein moieties allowing display areknown in the art and described herein, such as Aga2p and pIII.

The cell is preferably a bacterial cell or a eukaryotic cell, such as ayeast cell.

In a preferred embodiment, at least 2 of the nucleic acids of thepopulation encode different CDR3 amino acid sequences, in particulardifferent CDR-3H and/or CDR-3L amino acid sequences, more preferablywherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 or moreencode different CDR3 amino acid sequences, in particular differentCDR-3H and/or CDR-3L amino acid sequence, and/or wherein at least 50%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% of the nucleic acids of the populationencode different CDR3 amino acid sequences, in particular differentCDR-3H and/or CDR-3L amino acid sequences. In a more preferredembodiment the, sequences are diversified due to immunization of one ormore non-humans with a target of interest. Preferably, the sequences arediversified within the CDR3 amino acid sequence, in case the sequencefurther encompasses 1, 2, or 3 amino acids N-terminal and/or C-terminalof the non-human-derived CDR3 amino acid sequence.

A population of nucleic acids of the disclosure is particularly usefulfor mass humanization of non-human antibodies and subsequent screeningfor antibodies for suitable binding properties for an antigen ofinterest.

By expressing the population of nucleic acids in a suitable expressionsystem for display, a population of displayed, mass humanized proteins,in particular antibodies or antibody fragments such as scFv, Fv or Fabis obtained, which contain non-human CDR3 or non-human-derived CDR3regions.

In a further embodiment, the present disclosure provides a population ofproteins comprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences, obtainable by

(i) expressing at least one protein encoded by a population of thedisclosure above in a suitable expression system, and

(ii) displaying at least one protein on a virus, a cell or a surface.

Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 ormore proteins encoded by a population of the disclosure are expressed ina suitable expression system.

In a more preferred embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50,100, 150, 200 or more proteins encoded by a population of the disclosureare displayed on a virus, a cell or a surface, preferably wherein theproteins are fusion proteins, such as a fusion protein to a minor coatprotein of a bacterial phage or to Agap2p.

By expressing the population of nucleic acids in a suitable expressionsystem a population of mass humanized proteins, in particular antibodiesor antibody fragments such as scFv, Fv or Fab is obtained, which containnon-human CDR3 or non-human-derived CDR3 regions.

In some embodiments, the present disclosure provides a population ofproteins comprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences, wherein the human framework sequences comprise afirst human framework region (FR1), a second human framework region(FR2), a third human framework region (FR3), and a fourth humanframework region (FR4), such that the FR1 and FR2 regions areinterspaced by a complementarity determining region 1 (CDR1), the FR2and FR3 regions are interspaced by a complementarity determining region2 (CDR2), and the FR3 and FR4 regions are interspaced by anon-human-derived CDR3 amino acid sequence; wherein the CDR1 and CDR2amino acid sequences are diversified among the population of proteinscomprising at least one immunoglobulin variable domain, wherein eachCDR1 or CDR2 amino acid sequence is independently based (i) on a humanCDR1 or CDR2, respectively, or (ii) on a non-human CDR1 or CDR2,respectively; wherein at least some of the CDR1 or CDR2 amino acidsequences have been modified to comprise at least one amino acid presentin non-human CDR1 or CDR2 amino acid sequences, respectively, in case ofhuman CDR1 or CDR2, respectively, or to comprise at least one amino acidpresent in human CDR1 or CDR2 amino acid sequences, respectively, incase of non-human CDR1 or CDR2, respectively; and wherein the human FR1,FR2, FR3 and FR4 regions are human framework regions selected to providea scaffold conducive for non-human CDR3 amino acid sequences, with theproviso: that the two C-terminal amino acids of FR2 are optionallynon-human, and that the two C-terminal amino acids of FR3 are optionallynon-human.

The populations or libraries of the disclosure are particularly suitablefor mass humanization of non-human antibodies and allow for generalizingthe humanization process by providing scaffolds that represent thecodified landscape of all intermediate humanization across non-human andhuman with a population or library that explores the space between bothspecies.

In a preferred embodiment, the present disclosure provides a populationof proteins comprising at least one immunoglobulin variable domainhaving a non-human-derived CDR3 amino acid sequence embedded inessentially human framework sequences as described above, wherein the atleast one protein comprising at least one immunoglobulin variable domainhaving a non-human-derived CDR3 amino acid sequence embedded inessentially human framework sequences; comprises a VH domain, or a VLdomain, or a VH domain and a VL domain, or a heavy chain of an antibodyor a fragment thereof comprising the VH domain, and/or a light chain ofan antibody or a fragment thereof comprising the VL domain and/or anscFv, more preferably an scFv; and/or is selected from an immunoglobulinmolecule, a disulfide linked Fv, a monoclonal antibody, an scFv, asingle domain antibody, a diabody, a multispecific antibody, a Fab, aFab′, a bispecific antibody, a F(ab′)2, or an Fv, more preferably anscFv; and wherein the at least one protein optionally further comprisesat least one moiety allowing display of the at least one protein on avirus, a cell, or a surface.

In a more preferred embodiment, the at least one protein optionallyfurther comprises at least one moiety allowing display of the at leastone protein on a virus, a cell, or a surface. Such moieties allowingdisplay are described herein.

In a further preferred embodiment, the present disclosure provides apopulation of proteins comprising at least one immunoglobulin variabledomain having a non-human-derived CDR3 amino acid sequence embedded inessentially human framework sequences as described above, wherein the atleast one protein comprising at least one immunoglobulin variable domainhaving a non-human-derived CDR3 amino acid sequence embedded inessentially human framework sequences, wherein one or more, preferably1, 2, 3, or 4, different sets of framework regions comprising a firsthuman framework region (FR1), a second human framework region (FR2), athird human framework region (FR3), and a fourth human framework region(FR4) are present in the VH domains, and/or one or more, preferably 1,2, 3, or 4, different sets of framework regions comprising a first humanframework region (FR1), a second human framework region (FR2), a thirdhuman framework region (FR3), and a fourth human framework region (FR4)are present in the VL domains,

with the proviso:

that the two C-terminal amino acids of FR2 are optionally non-human, and

that the two C-terminal amino acids of FR3 are optionally non-human.

It is understood that the preferred embodiments for the methods of thedisclosure also apply to the populations of proteins described herein.

In particular, the human FR1, FR2, FR3 and FR4 regions are humanframework regions with the proviso that the two C-terminal amino acidsof FR2 are optionally non-human, and that the two C-terminal amino acidsof FR3 are optionally non-human.

Human FR1, FR2, FR3 and FR4 regions are FR1, FR2, FR3 and FR4 regionswhich are naturally occurring in humans. As described above, the twoC-terminal amino acids of FR2 are optionally non-human, and that the twoC-terminal amino acids of FR3 are optionally non-human. In someembodiments, the FR1 and FR4 regions are human FR1 and FR4 regions. Asalso described above, it is possible that a non-human-derived CDR3 aminoacid sequence, preferably a non-human-CDR3 amino acid sequence, furthercomprising 1, 2, or 3 amino acids N-terminal of the non-human-derivedCDR3 amino acid sequence, and/or 1, 2, or 3 amino acids C-terminal ofthe non-human-derived CDR3 amino acid sequence is embedded, inparticular in case a non-human SDR is embedded. For example, an SDRcomprising the CDR-3H may be embedded. In such embodiment, the followingfurther N-terminal and C-terminal amino acids, respectively, arepresent: XXXXXXXXXXDY| W (SEQ ID NO: 1).

Therefore, in another preferred embodiment, at least one non-human CDR3sequence is embedded, such that the human FR3 and FR4 regions areinterspaced by a non-human CDR3 amino acid sequence. Preferably, alsothe resulting FR3 and FR4 regions will be human.

Further, FR2 is a human FR2 framework region, or the two C-terminalamino acids of FR2 are optionally non-human. In one preferredembodiment, FR2 is a human FR2 framework region.

In another preferred embodiment, the two C-terminal amino acids of FR2are non-human, whereas the remaining sequence of FR2 is human.

In a further preferred embodiment, the proteins of the populationcomprise at least one VH domain and/or at least one VL domain, morepreferably the proteins comprise or represent an antibody, an scFv, a Fvor Fab.

The CDR1 and CDR2 amino acid sequences are diversified among thepopulation of proteins comprising at least one immunoglobulin variabledomain, wherein each CDR1 or CDR2 amino acid sequence is independentlybased i) on a human CDR1 or CDR2, respectively, or ii) on a non-humanCDR1 or CDR2, respectively, wherein at least some of the CDR1 or CDR2amino acid sequences have been modified to contain at least one aminoacid present in non-human CDR1 or CDR2 amino acid sequences,respectively, in case of human CDR1 or CDR2, respectively, or to containat least one amino acid present in human CDR1 or CDR2 amino acidsequences, respectively, in case of non-human CDR1 or CDR2,respectively.

Diversified CDR1 and CDR2 sequences according to the disclosure can beobtained by computationally integrating a plurality, such as 10⁴, 10⁵,or 10⁶-10⁷ of simulated humanizations across the non-human antibodyrepertoire and the human acceptor antibody repertoire, in order togenerate a mathematically optimal Bayesian representation of thehumanization space between non-human and human, converting theprobabilistic model to a frequentist interpretation, thereby obtaining apopulation or library. Each position ends up containing a probability ofencountering the human germline residue, the non-human germlineresidues, and the most common collection of affinity maturation residuesfrom both species.

In a preferred embodiment, cysteine, and/or methionine residues areremoved and/or the asparagine residue content is reduced, as theseresidues form biochemical liabilities.

In a yet further preferred embodiment, the human or non-human CDR1regions and the human and non-human CDR2 regions, on which thediversified CDR1 and CDR2 amino acid sequences are based, are selectedfrom human germline CDR1 regions, human germline CDR2 regions, non-humangermline CDR1 regions, non-human germline CDR2 regions, human somatichypermutation CDR1 regions, human somatic hypermutation CDR2 regions,non-human somatic hypermutation CDR1 regions, non-human somatichypermutation CDR2 regions, non-human gene conversion CDR1 regions, andnon-human gene conversion CDR2 regions.

In a yet further preferred embodiment, the diversified CDR1 and CDR2amino acid sequences are CDR-H1, CDR-H2, CDR-L1 and/or CDR-L2 sequences.

In a preferred embodiment, the population includes at least one humanCDR-H1, and/or a human CDR-H2, and/or a human CDR-L1 and/or a humanCDR-L2 sequence.

In a further preferred embodiment, the population includes at least onenon-human CDR-H1, and/or a non-human CDR-H2, and/or a non-human CDR-L1and/or a non-human CDR-L2 sequence.

In some embodiments, the human FR1, FR2, FR3 and FR4 regions are humanframework regions selected to provide a scaffold conducive for non-humanCDR3 amino acid sequences, with the proviso that the two C-terminalamino acids of FR2 are optionally non-human, and that the two C-terminalamino acids of FR3 are optionally non-human. In one preferredembodiment, the population of the disclosure comprises at least oneprotein comprising a human FR1, a human FR2, a human FR3 and a human FR4region, and a human CDR1, and a human CDR2 region.

In another preferred embodiment, the population of the disclosurecomprises at least one protein comprising a human FR1, a human FR2, ahuman FR3 and a human FR4 region, and a non-human CDR1, and a non-humanCDR2 region.

In yet another preferred embodiment, the population of the disclosuredoes not contain a protein comprising a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a human CDR1, and a human CDR2 region.

In yet another preferred embodiment, the population of the disclosuredoes not contain a protein comprising a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a non-human CDR1, and a non-human CDR2region.

A suitable scaffold conducive for non-human CDR3 amino acid sequences isobtainable by selecting a framework set by performing the followingsteps:

computational imputation of germline repertoire element centroids whichare most utilized in functional non-human antibodies

generating an amino acid alignment of human frameworks compared to saidcomputationally imputed non-human frameworks and selecting the closestrepresentatives, respectively, that have the same length of CDR-H1,CDR-H2, CDR-L1, and CDR-L2 and similar sequence composition,respectively,

further reducing the number of candidates framework sequences bypreferring templates that are known to be stable in the art andoptionally have worked as previous drugs,

further reducing the number of framework candidates by structurallymodeling antibodies from non-human and human using these frameworks andanalyzing their structural superposition tolerance by root mean squareddeviation (RMSD), and

selecting a set of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heavy chain and 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 light chain frameworks comprising set of FR1,FR2, FR3 and FR4 regions, respectively.

For example, a set of 2 heavy chain and 2 light chain frameworkscomprising set of FR1, FR2, FR3 and FR4 regions respectively, can bepresent. Moreover, the two C-terminal amino acids of heavy chain FR2 mayoptionally be non-human, and the two C-terminal amino acids of heavychain FR3 may optionally be non-human, for example in case an SDR isgrafted into a scaffold, or in case amino acids surrounding FR2 aresubstituted, as described above.

In a yet further preferred embodiment, the human FR1, FR2, FR3 and FR4regions which are human framework regions selected to provide a scaffoldconducive for non-human CDR3 amino acid sequences are obtainable by:

(i) providing

(a) a collection of sequences of naturally occurring human antibodieseach comprising a set of human FR1, FR2, FR3 and FR4 regions; and

(b) a collection of sequences of naturally occurring non-humanantibodies each comprising a set of non-human FR1, FR2, FR3 and FR4regions, and

(ii) identifying a plurality of sets of human FR1, FR2, FR3 and FR4regions which provide a scaffold conducive for non-human CDR3 amino acidsequences by

determining the parameters framework homology, CDR cosmology, CDRlengths, CDR canonical structure, and adoption of comfortable heavy orlight mount angles, and

selecting sets of human FR1, FR2, FR3 and FR4 regions which exhibit highscores for the parameters,

and/or

the two C-terminal amino acids of heavy chain FR2 are optionallynon-human, and

the two C-terminal amino acids of heavy chain FR3 are optionallynon-human.

In a preferred embodiment, at least 2 of the proteins of the populationcomprise different CDR3 amino acid sequences, in particular differentCDR-3H and/or CDR-3L amino acid sequences,

more preferably wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100,150, 200 or more proteins comprise different CDR3 amino acid sequences,in particular different CDR-3H and/or CDR-3L amino acid sequence, and/or

wherein at least 50%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% of the proteinscomprise different CDR3 amino acid sequences, in particular differentCDR-3H and/or CDR-3L amino acid sequences. In a more preferredembodiment, the sequences are diversified due to immunization of one ormore non-humans with a target of interest. Preferably, the sequences arediversified within the CDR3 amino acid sequence, in case the sequencefurther encompasses 1, 2, or 3 amino acids N-terminal and/or C-terminalof the non-human-derived CDR3 amino acid sequence.

A population of proteins of the disclosure is particularly useful formass humanization of non-human antibodies and subsequent screening forantibodies for suitable binding properties for an antigen of interest.

Further, the disclosure provides for Acceptor framework libraries, whichare suitable for methods and uses of the present disclosure.

In a further embodiment, the present disclosure provides a population ofAcceptor Framework nucleic acid, wherein each Acceptor Framework nucleicacid comprises nucleic acids encoding a set of framework regionscomprising a first human framework region (FR1), a second humanframework region (FR2), a third human framework region (FR3), and afourth human framework region (FR4); wherein the FR1 and FR2 regions areinterspaced by a complementarity determining region 1 (CDR1), the FR2and FR3 regions are interspaced by a complementarity determining region2 (CDR2), and the nucleic acid sequences encoding FR3 and FR4 regionsare linked directly or are interspaced by a stuffer nucleic acidsequence; and wherein the nucleic acid sequences encoding the CDR1 andCDR2 amino acid sequences are diversified among the population ofnucleic acids encoding at least one protein comprising at least oneimmunoglobulin variable domain, wherein each nucleic acid sequenceencoding a CDR1 or CDR2 amino acid sequence is independently based (i)on a nucleic acid sequence encoding a human CDR1 or CDR2, respectively,or (ii) on a nucleic acid sequence encoding a non-human CDR1 or CDR2,respectively; wherein at least some of the nucleic acid sequencesencoding a CDR1 or CDR2 amino acid sequence have been modified to encodeat least one amino acid present in non-human CDR1 or CDR2 amino acidsequences, respectively, in case of human CDR1 or CDR2, respectively, orto encode at least one amino acid present in human CDR1 or CDR2 aminoacid sequences, respectively, in case of non-human CDR1 or CDR2,respectively; and wherein the human FR1, FR2, FR3 and FR4 regions arehuman framework regions selected to provide a scaffold conducive fornon-human CDR3 amino acid sequences, with the proviso: that the twoC-terminal amino acids of FR2 are optionally non-human, and that the twoC-terminal amino acids of FR3 are optionally non-human.

It is understood that the preferred embodiments for the methods of thedisclosure and populations of the disclosure also apply to thepopulations of Acceptor Framework nucleic acids described herein.

In particular, the human FR1, FR2, FR3 and FR4 regions are humanframework regions with the proviso that the two C-terminal amino acidsof FR2 are optionally non-human, and that the two C-terminal amino acidsof FR3 are optionally non-human.

Human FR1, FR2, FR3 and FR4 regions are FR1, FR2, FR3 and FR4 regionswhich are naturally occurring in humans. As described above, the twoC-terminal amino acids of FR2 are optionally non-human, and that the twoC-terminal amino acids of FR3 are optionally non-human. In someembodiments, the FR1 and FR4 regions are human FR1 and FR4 regions.Preferably, the FR3 is human.

Further, FR2 is a human FR2 framework region, or the two C-terminalamino acids of FR2 are optionally non-human. In one preferredembodiment, FR2 is a human FR2 framework region.

In another preferred embodiment, the two C-terminal amino acids of FR2are non-human, whereas the remaining sequence of FR2 is human.

The nucleic acids encoding the CDR1 and CDR2 amino acid sequences arediversified among the population of Acceptor Framework nucleic acids,wherein each nucleic acid sequence encoding a CDR1 or CDR2 amino acidsequence is independently based i) on a nucleic acid sequence encoding ahuman CDR1 or CDR2, respectively, or ii) on a nucleic acid sequenceencoding a non-human CDR1 or CDR2, respectively, wherein at least someof the nucleic acid sequences encoding a CDR1 or CDR2 amino acidsequence have been modified to encode at least one amino acid present innon-human CDR1 or CDR2 amino acid sequences, respectively, in case ofhuman CDR1 or CDR2, respectively, or to encode at least one amino acidpresent in human CDR1 or CDR2 amino acid sequences, respectively, incase of non-human CDR1 or CDR2, respectively.

Diversified CDR1 and CDR2 sequences according to the disclosure can beobtained by computationally integrating a plurality, such as 10⁴, 10⁵,or 10⁶-10⁷ of simulated humanizations across the non-human antibodyrepertoire and the human acceptor antibody repertoire, in order togenerate a mathematically optimal Bayesian representation of thehumanization space between non-human and human, converting theprobabilistic model to a frequentist interpretation, thereby obtaining apopulation or library. Each position ends up containing a probability ofencountering the human germline residue, the non-human germlineresidues, and the most common collection of affinity maturation residuesfrom both species.

In a preferred embodiment, cysteine, and/or methionine residues areremoved and/or the asparagine residue content is reduced, as theseresidues form biochemical liabilities.

In a yet further preferred embodiment, the human or non-human CDR1regions and the human and non-human CDR2 regions, on which thediversified CDR1 and CDR2 amino acid sequences are based, are selectedfrom human germline CDR1 regions, human germline CDR2 regions, non-humangermline CDR1 regions, non-human germline CDR2 regions, human somatichypermutation CDR1 regions, human somatic hypermutation CDR2 regions,non-human somatic hypermutation CDR1 regions, non-human somatichypermutation CDR2 regions, non-human gene conversion CDR1 regions, andnon-human gene conversion CDR2 regions.

In a yet further preferred embodiment, the diversified CDR1 and CDR2amino acid sequences are CDR-H1, CDR-H2, CDR-L1 and/or CDR-L2 sequences.

In a preferred embodiment, the population includes at least one nucleicacid encoding a human CDR-H1, and/or a human CDR-H2, and/or a humanCDR-L1 and/or a human CDR-L2 sequence.

In a further preferred embodiment, the population includes at least onenucleic acid encoding a non-human CDR-H1, and/or a non-human CDR-H2,and/or a non-human CDR-L1 and/or a non-human CDR-L2 sequence.

In some embodiments, the human FR1, FR2, FR3 and FR4 regions are humanframework regions selected to provide a scaffold conducive for non-humanCDR3 amino acid sequences, with the proviso that the two C-terminalamino acids of FR2 are optionally non-human, and that the two C-terminalamino acids of FR3 are optionally non-human. In one preferredembodiment, a population of the disclosure comprises at least onenucleic acid encoding a human FR1, a human FR2, a human FR3 and a humanFR4 region, and a human CDR1, and a human CDR2 region.

In another preferred embodiment, a population of the disclosurecomprises at least one nucleic acid encoding a human FR1, a human FR2, ahuman FR3 and a human FR4 region, and a non-human CDR1, and a non-humanCDR2 region.

In yet another preferred embodiment, a population of the disclosure doesnot contain a nucleic acid encoding a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a human CDR1, and a human CDR2 region.

In yet another preferred embodiment, a population of the disclosure doesnot contain a nucleic acid encoding a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a non-human CDR1, and a non-human CDR2region.

A suitable scaffold conducive for non-human CDR3 amino acid sequences isobtainable by selecting a framework set by performing the followingsteps:

computational imputation of germline repertoire element centroids whichare most utilized in functional non-human antibodies

generating an amino acid alignment of human frameworks compared to saidcomputationally imputed non-human frameworks and selecting the closestrepresentatives, respectively, that have the same length of CDR-H1,CDR-H2, CDR-L1, and CDR-L2 and similar sequence composition,respectively,

further reducing the number of candidates framework sequences bypreferring templates that are known to be stable in the art andoptionally have worked as previous drugs,

further reducing the number of framework candidates by structurallymodeling antibodies from non-human and human using these frameworks andanalyzing their structural superposition tolerance by root mean squareddeviation (RMSD), and

selecting a set of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heavy chain and 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 light chain frameworks comprising set of FR1,FR2, FR3 and FR4 regions, respectively.

As an example, a set of 2 heavy chain and 2 light chain frameworkscomprising set of FR1, FR2, FR3 and FR4 regions respectively, can bepresent. Moreover, the two C-terminal amino acids of heavy chain FR2 mayoptionally be non-human, and the two C-terminal amino acids of heavychain FR3 may optionally be non-human, for example in case an SDR isgrafted into a scaffold, or in case amino acids surrounding FR2 aresubstituted, as described above.

In a yet further preferred embodiment, the human FR1, FR2, FR3 and FR4regions which are human framework regions selected to provide a scaffoldconducive for non-human CDR3 amino acid sequences are obtainable by:

(i) providing

(a) a collection of sequences of naturally occurring human antibodieseach comprising a set of human FR1, FR2, FR3 and FR4 regions; and

(b) a collection of sequences of naturally occurring non-humanantibodies each comprising a set of non-human FR1, FR2, FR3 and FR4regions, and

(ii) identifying a plurality of sets of human FR1, FR2, FR3 and FR4regions which provide a scaffold conducive for non-human CDR3 amino acidsequences by

determining the parameters framework homology, CDR cosmology, CDRlengths, CDR canonical structure, and adoption of comfortable heavy orlight mount angles, and

selecting sets of human FR1, FR2, FR3 and FR4 regions which exhibit highscores for the parameters,

and/or

the two C-terminal amino acids of heavy chain FR2 are optionallynon-human, and

the two C-terminal amino acids of heavy chain FR3 are optionallynon-human.

In a further embodiment, the present disclosure provides a population ofAcceptor Framework proteins, wherein each Acceptor Framework proteincomprises a set of framework regions comprising a first human frameworkregion (FR1), a second human framework region (FR2), a third humanframework region (FR3), and a fourth human framework region (FR4);wherein the FR1 and FR2 regions are interspaced by a complementaritydetermining region 1 (CDR1), the FR2 and FR3 regions are interspaced bya complementarity determining region 2 (CDR2), and the FR3 and FR4regions are linked directly or are interspaced by a stuffer sequence;and wherein the CDR1 and CDR2 amino acid sequences are diversified amongthe population of Acceptor Framework proteins, wherein each CDR1 or CDR2amino acid sequence is independently based (i) on a human CDR1 or CDR2,respectively, or (ii) on a non-human CDR1 or CDR2, respectively, whereinat least some of the CDR1 or CDR2 amino acid sequence have been modifiedto comprise at least one amino acid present in non-human CDR1 or CDR2amino acid sequences, respectively, in case of human CDR1 or CDR2,respectively, or to comprise at least one amino acid present in humanCDR1 or CDR2 amino acid sequences, respectively, in case of non-humanCDR1 or CDR2, respectively; and wherein the human FR1, FR2, FR3 and FR4regions are human framework regions selected to provide a scaffoldconducive for non-human CDR3 amino acid sequences, with the proviso:

that the two C-terminal amino acids of FR2 are optionally non-human, and

that the two C-terminal amino acids of FR3 are optionally non-human.

It is understood that the preferred embodiments for the methods of thedisclosure and populations of the disclosure also apply to thepopulations of Acceptor Framework proteins described herein.

In particular, the human FR1, FR2, FR3 and FR4 regions are humanframework regions with the proviso that the two C-terminal amino acidsof FR2 are optionally non-human, and that the two C-terminal amino acidsof FR3 are optionally non-human.

Human FR1, FR2, FR3 and FR4 regions are FR1, FR2, FR3 and FR4 regionswhich are naturally occurring in humans. As described above, the twoC-terminal amino acids of FR2 are optionally non-human, and that the twoC-terminal amino acids of FR3 are optionally non-human. In someembodiments, the FR1 and FR4 regions are human FR1 and FR4 regions.Preferably, also the FR3 and FR4 region is human.

Further, FR2 is a human FR2 framework region, or the two C-terminalamino acids of FR2 are optionally non-human. In one preferredembodiment, FR2 is a human FR2 framework region.

In another preferred embodiment, the two C-terminal amino acids of FR2are non-human, whereas the remaining sequence of FR2 is human.

The CDR1 and CDR2 amino acid sequences are diversified among thepopulation of Acceptor Framework proteins, wherein each CDR1 or CDR2amino acid sequence is independently based i) on a human CDR1 or CDR2,respectively, or ii) on a non-human CDR1 or CDR2, respectively, whereinat least some of the CDR1 or CDR2 amino acid sequences have beenmodified to contain at least one amino acid present in non-human CDR1 orCDR2 amino acid sequences, respectively, in case of human CDR1 or CDR2,respectively, or to contain at least one amino acid present in humanCDR1 or CDR2 amino acid sequences, respectively, in case of non-humanCDR1 or CDR2, respectively.

Diversified CDR1 and CDR2 sequences according to the disclosure can beobtained by computationally integrating a plurality, such as 10⁴, 10⁵,or 10⁶-10⁷ of simulated humanizations across the non-human antibodyrepertoire and the human acceptor antibody repertoire, in order togenerate a mathematically optimal Bayesian representation of thehumanization space between non-human and human, converting theprobabilistic model to a frequentist interpretation, thereby obtaining apopulation or library. Each position ends up containing a probability ofencountering the human germline residue, the non-human germlineresidues, and the most common collection of affinity maturation residuesfrom both species.

In a preferred embodiment, cysteine, and/or methionine residues areremoved and/or the asparagine residue content is reduced, as theseresidues form biochemical liabilities.

In a yet further preferred embodiment, the human or non-human CDR1regions and the human and non-human CDR2 regions, on which thediversified CDR1 and CDR2 amino acid sequences are based, are selectedfrom human germline CDR1 regions, human germline CDR2 regions, non-humangermline CDR1 regions, non-human germline CDR2 regions, human somatichypermutation CDR1 regions, human somatic hypermutation CDR2 regions,non-human somatic hypermutation CDR1 regions, non-human somatichypermutation CDR2 regions, non-human gene conversion CDR1 regions, andnon-human gene conversion CDR2 regions.

In a yet further preferred embodiment, the diversified CDR1 and CDR2amino acid sequences are CDR-H1, CDR-H2, CDR-L1 and/or CDR-L2 sequences.

In a preferred embodiment, the population of Acceptor Framework proteinsincludes at least one human CDR-H1, and/or a human CDR-H2, and/or ahuman CDR-L1 and/or a human CDR-L2 sequence.

In a further preferred embodiment, the population of Acceptor Frameworkproteins includes at least one non-human CDR-H1, and/or a non-humanCDR-H2, and/or a non-human CDR-L1 and/or a non-human CDR-L2 sequence.

In some embodiments, the human FR1, FR2, FR3 and FR4 regions are humanframework regions selected to provide a scaffold conducive for non-humanCDR3 amino acid sequences, with the proviso that the two C-terminalamino acids of FR2 are optionally non-human, and that the two C-terminalamino acids of FR3 are optionally non-human. In one preferredembodiment, a population of the disclosure comprises at least oneprotein comprising a human FR1, a human FR2, a human FR3 and a human FR4region, and a human CDR1, and a human CDR2 region.

In another preferred embodiment, a population of the disclosurecomprises at least one protein comprising a human FR1, a human FR2, ahuman FR3 and a human FR4 region, and a non-human CDR1, and a non-humanCDR2 region.

In yet another preferred embodiment, the population of the disclosuredoes not contain a protein comprising a human FR1, a human FR2, a humanFR3 and a human FR4 region, and a human CDR1, and a human CDR2 region.

In yet another preferred embodiment, a population of the disclosure doesnot contain a protein comprising a human FR1, a human FR2, a human FR3and a human FR4 region, and a non-human CDR1, and a non-human CDR2region.

A suitable scaffold conducive for non-human CDR3 amino acid sequences isobtainable by selecting a framework set by performing the followingsteps:

computational imputation of germline repertoire element centroids whichare most utilized in functional non-human antibodies

generating an amino acid alignment of human frameworks compared to saidcomputationally imputed non-human frameworks and selecting the closestrepresentatives, respectively, that have the same length of CDR-H1,CDR-H2, CDR-L1, and CDR-L2 and similar sequence composition,respectively,

further reducing the number of candidates framework sequences bypreferring templates that are known to be stable in the art andoptionally have worked as previous drugs,

further reducing the number of framework candidates by structurallymodeling antibodies from non-human and human using these frameworks andanalyzing their structural superposition tolerance by root mean squareddeviation (RMSD), and

selecting a set of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heavy chain and 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 light chain frameworks comprising set of FR1,FR2, FR3 and FR4 regions, respectively.

Moreover, the two C-terminal amino acids of heavy chain FR2 areoptionally non-human, and the two C-terminal amino acids of heavy chainFR3 are optionally non-human, for example in case an SDR is grafted intoa scaffold, or in case amino acids surrounding FR2 are substituted, asdescribed above.

In a yet further preferred embodiment, the human FR1, FR2, FR3 and FR4regions which are human framework regions selected to provide a scaffoldconducive for non-human CDR3 amino acid sequences are obtainable by:

(i) providing

(a) a collection of sequences of naturally occurring human antibodieseach comprising a set of human FR1, FR2, FR3 and FR4 regions; and

(b) a collection of sequences of naturally occurring non-humanantibodies each comprising a set of non-human FR1, FR2, FR3 and FR4regions, and

(ii) identifying a plurality of sets of human FR1, FR2, FR3 and FR4regions which provide a scaffold conducive for non-human CDR3 amino acidsequences by

determining the parameters framework homology, CDR cosmology, CDRlengths, CDR canonical structure, and adoption of comfortable heavy orlight mount angles, and

selecting sets of human FR1, FR2, FR3 and FR4 regions which exhibit highscores for the parameters,

and/or

the two C-terminal amino acids of heavy chain FR2 are optionallynon-human, and

the two C-terminal amino acids of heavy chain FR3 are optionallynon-human.

In a further embodiment, the present disclosure provides the use of apopulation of nucleic acids of the disclosure, or a population ofproteins of the disclosure, for screening for proteins comprising atleast one immunoglobulin variable domain, in particular antibodies orfragments thereof, which specifically bind to an antigen of interest.Preferably, the fragment of an antibody comprises at least oneimmunoglobulin variable domain, in a more preferred embodiment, thefragment of an antibody is a Fv, scFv or Fab.

Methods for screening via display methods are described in detail above.

A protein comprising at least one immunoglobulin variable domain, inparticular an antibody or fragment thereof, is understood tospecifically bind to an antigen when the protein binds to the antigen,preferably binding with an affinity of Kd of 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ orless, and substantially does not bind to other polypeptides or binds toother polypeptides with at least 10-fold or at least 100-fold weakeraffinity, preferably with a Kd of 10⁻⁶, 10⁻⁵, or more.

The strength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (Kd) of the interaction,wherein a smaller Kd represents a greater affinity. The Kd value can bedetermined by methods known in the art, such as at 25° C. by surfaceplasmon resonance spectroscopy. Systems for performing such analyses arecommercially available (e.g. using a Biacore3000™ surface plasmonresonance (SPR) system, Biacore, INC, Piscataway N.J.). Kineticassociation rates (kon) and dissociation rates (koff) can be obtainedsimultaneously by fitting the data globally to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (KD) values can be calculated as koff/kon. Bindingaffinity of an antibody is generally measured at 25° C., but can also bemeasured at 37° C.

In some embodiments of any of the various the methods and uses describedherein, screening is performed by display of at least one protein on avirus, a cell, or a surface.

In any of the various methods described herein in which it may beadvantageously employed, a method may comprise extraction of nucleicacid from a sample. Methods for the extraction and purification ofnucleic acids are well known in the art. For example, nucleic acids canbe purified by organic extraction with phenol, phenol/chloroform/isoamylalcohol, or similar formulations, including TRIzol and TriReagent. Othernon-limiting examples of extraction techniques include: (1) organicextraction followed by ethanol precipitation, e.g., using aphenol/chloroform organic reagent with or without the use of anautomated nucleic acid extractor, e.g., the Model 341 DNA Extractoravailable from Applied Biosystems (Foster City, Calif.); (2) stationaryphase adsorption methods; and (3) salt-induced nucleic acidprecipitation methods, such precipitation methods being typicallyreferred to as “salting-out” methods. Another example of nucleic acidisolation and/or purification includes the use of magnetic particles towhich nucleic acids can specifically or non-specifically bind, followedby isolation of the beads using a magnet, and washing and eluting thenucleic acids from the beads. In some embodiments, the above isolationmethods may be preceded by an enzyme digestion step to help eliminateunwanted protein from the sample, e.g., digestion with proteinase K, orother like proteases. If desired, RNase inhibitors may be added to thelysis buffer. For certain cell or sample types, it may be desirable toadd a protein denaturation/digestion step to the protocol. Purificationmethods may be directed to isolate DNA, RNA, or both. When both DNA andRNA are isolated together during or subsequent to an extractionprocedure, further steps may be employed to purify one or bothseparately from the other. Sub-fractions of extracted nucleic acids canalso be generated, for example, purification by size, sequence, or otherphysical or chemical.

The extracted polynucleotides from the samples can be sequenced togenerate sequencing reads. A variety of suitable sequencing technologiesare available. Exemplary sequencing techniques can include, for exampleemulsion PCR (pyrosequencing from Roche 454, semiconductor sequencingfrom Ion Torrent, SOLiD sequencing by ligation from Life Technologies,sequencing by synthesis from Intelligent Biosystems), bridgeamplification on a flow cell (e.g. Solexa/lllumina), isothermalamplification by Wildfire technology (Life Technologies) orrolonies/nanoballs generated by rolling circle amplification (CompleteGenomics, Intelligent Biosystems, Polonator). Sequencing technologieslike Heliscope (Helicos), SMRT technology (Pacific Biosciences) ornanopore sequencing (Oxford Nanopore) allow direct sequencing of singlemolecules without prior clonal amplification may be suitable sequencingplatforms. Polynucleotides from a sample may be amplified by anysuitable means prior to and/or during sequencing.

In any of the various methods described herein in which it may beadvantageously employed, a method may comprise a polynucleotideamplification reaction. In general, “polynucleotide amplification”refers to a process by which one or more copies of a polynucleotide aregenerated. A variety of suitable amplification processes are available.Amplification methods may involve changes in temperature, such as a heatdenaturation step, or may be isothermal processes that do not requireheat denaturation. The polymerase chain reaction (PCR) uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of thetarget sequence. Denaturation of annealed nucleic acid strands may beachieved by the application of heat, increasing local metal ionconcentrations (e.g. U.S. Pat. No. 6,277,605), ultrasound radiation(e.g. WO/2000/049176), application of voltage (e.g. U.S. Pat. Nos.5,527,670, 6,033,850, 5,939,291, and 6,333,157), and application of anelectromagnetic field in combination with primers bound to amagnetically-responsive material (e.g. U.S. Pat. No. 5,545,540). In avariation called RT-PCR, reverse transcriptase (RT) is used to make acomplementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCRto produce multiple copies of DNA (e.g. U.S. Pat. Nos. 5,322,770 and5,310,652). Other amplification methods include rolling circleamplification (RCA) (e.g., Lizardi, “Rolling Circle Replication ReporterSystems,” U.S. Pat. No. 5,854,033); helicase dependent amplification(HDA) (e.g., Kong et al., “Helicase Dependent Amplification NucleicAcids,” U.S. Pat. Appln. Pub. No. US 2004-0058378 A1); and loop-mediatedisothermal amplification (LAMP) (e.g., Notomi et al., “Process forSynthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278). In some cases,isothermal amplification utilizes transcription by an RNA polymerasefrom a promoter sequence, such as may be incorporated into anoligonucleotide primer. Transcription-based amplification methodscommonly used in the art include nucleic acid sequence basedamplification, also referred to as NASBA (e.g. U.S. Pat. No. 5,130,238);methods which rely on the use of an RNA replicase to amplify the probemolecule itself, commonly referred to as Qβ replicase (e.g., Lizardi, P.et al. (1988) BioTechnol. 6, 1197-1202); self-sustained sequencereplication (e.g., Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci. USA87, 1874-1878; Landgren (1993) Trends in Genetics 9, 199-202; and HELENH. LEE et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)); andmethods for generating additional transcription templates (e.g. U.S.Pat. Nos. 5,480,784 and 5,399,491). Isothermal amplification processescan be linear or exponential.

In one aspect, the disclosure provides systems for performing any of themethods described herein. For example, the system may comprise one ormore computer processors programmed to perform one or more steps of amethod described herein. Processors may be associated with one or morecontrollers, calculation units, and/or other units of a computer system,or implanted in firmware as desired. If implemented in software, theroutines may be stored in any computer readable memory such as in RAM,ROM, flash memory, a magnetic disk, a laser disk, or other storagemedium. Likewise, this software may be delivered to a computing devicevia any known delivery method including, for example, over acommunication channel such as a telephone line, the internet, a wirelessconnection, etc., or via a transportable medium, such as a computerreadable disk, flash drive, etc. The various steps may be implemented asvarious blocks, operations, tools, modules or techniques which, in turn,may be implemented in hardware, firmware, software, or any combinationthereof. When implemented in hardware, some or all of the blocks,operations, techniques, etc. may be implemented in, for example, acustom integrated circuit (IC), an application specific integratedcircuit (ASIC), a field programmable logic array (FPGA), a programmablelogic array (PLA), etc. In some embodiments, the computer is configuredto perform one or more steps of a method in response to a user request.The computer may receive the user request directly (e.g. by way of aninput device such as a keyboard, mouse, or touch screen operated by thecustomer or a user entering a customer request) or indirectly (e.g.through a wired or wireless connection, including over the internet).

In one aspect, the disclosure provides a computer-readable mediumcomprising codes that, upon execution by one or more processors,implements a method according to any of the methods disclosed herein.Computer readable medium may take many forms, including but not limitedto, a tangible storage medium, a carrier wave medium, or physicaltransmission medium. Non-volatile storage media include, for example,optical or magnetic disks, such as any of the storage devices in anycomputer(s) or the like, such as may be used to implement thecalculation steps, processing steps, etc. Volatile storage media includedynamic memory, such as main memory of a computer. Tangible transmissionmedia include coaxial cables; copper wire and fiber optics, includingthe wires that comprise a bus within a computer system. Carrier-wavetransmission media can take the form of electric or electromagneticsignals, or acoustic or light waves such as those generated during radiofrequency (RF) and infrared (IR) data communications. Common forms ofcomputer-readable media therefore include for example: a floppy disk, aflexible disk, hard disk, magnetic tape, any other magnetic medium, aCD-ROM, DVD or DVD-ROM, any other optical medium, punch cards papertape, any other physical storage medium with patterns of holes, a RAM, aPROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, acarrier wave transporting data or instructions, cables or linkstransporting such a carrier wave, or any other medium from which acomputer can read programming code and/or data. Many of these forms ofcomputer readable media may be involved in carrying one or moresequences of one or more instructions to a processor for execution.

In one aspect, the present disclosure provides for use of a compositionin the preparation for a medicament for the treatment of a disease orcondition of a subject. The compositions may comprise one or moreproteins or nucleic acids disclosed herein. In another aspect, thedisclosure provides methods of treating a subject for a disease orcondition, comprising administering to a subject one or more proteins ornucleic acids disclosed herein, or compositions thereof. Thecompositions may further comprise one or more pharmaceuticallyacceptable salts, excipients or vehicles. Compositions may be suitablefor various routes of administration, including, but not limited to,parenteral, intraarticular, subcutaneous, intravenous, intramuscular,intraperitoneal, intracerebral, intracerebroventricular, intramuscular,intraocular, intraarterial, or intralesional administration. The diseaseor condition, by way of non-limiting example, may be selected from thegroup consisting of an autoimmune disease, a cancer, a metabolicdisorder, a cardiovascular condition, a neurological condition, aneuromuscular condition, and an infection. In some embodiments, thetherapeutic composition comprises one or more antibodies humanized inaccordance with a method of the present disclosure. The specific diseaseor condition may depend on the therapeutic target, such as the target towhich the antibody is directed.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: The Generation of Humanized Antibodies

The generation of humanized antibodies can performed by the followingsteps:

(a) Immunization of a non-human species.

(b) Lymphocyte preparation from different organs (e.g. spleen, bonemarrow, or blood).

(c) RNA isolation from lymphocytes.

(d) Separate PCRs of non-human VH and VL variable regions.

(e) Separate nested PCRs of non-human VH and VL CDR3.

(f) Cloning of VH and/or VL CDR3s into acceptor framework libraries togenerate humanized VH and VL fragments.

(g) Cloning of obtained humanized heavy chain, humanized light chains,or humanized scFv into a phage display vector.

(h) Selection for specificity on antigen.

(i) Characterization of individual antibodies.

(j) Optimization based on characterization of successful antibodies andless successful antibodies.

In order to generate the appropriate Acceptor Frameworks, the non-humanV-gene scaffolds are compared to their human counterparts to identifyhuman V-genes that are (1) similar at the amino acid level, defined bypercent identity by amino acid alignment or similarity as defined by theBLOSUM62 similarity matrix, or some other preferred similarity matrix;(2) optionally possess the same length CDR1 and CDR2 sequences or mostsimilar length CDR1 and CDR2 sequences as the non-human V-gene; (3) havesimilar canonical classifications of their CDR1 and CDR2 structures(e.g. as determined by crystal structure or as predicted by amino acidmotif as determined by non-limiting, exemplary methods set forth inAl-Lazikani, B. et al. Standard conformations for the canonicalstructures of immunoglobulins. Journal of Molecular Biology 273, 927-948(1997)); and (4) are observed by crystal structure or predicted bycomputational modeling to adopt a similar H/L interface mount angle asdetermined by predicted or observed crystal structures showing thedegree shift of the central axis of the light chain Fv compared to afixed superposition of heavy chain Fv as described earlier. The numberof potential humanization frameworks can be further reduced topreferentially choose frameworks that have been previously used in humantherapeutics and are known to be stable (e.g. IGHV3 and IGHV1 familiesof the heavy chain V-gene). The subset of human frameworks that fitthese criteria is referred to in this example as “preferred acceptorscaffolds.”

A map is generated between every observed antibody in the non-humanspecies and every preferred acceptor scaffold in humans. All sequencesfrom both species are analyzed to: (1) obtain an optionallynon-redundant database of amino acid sequences from both species from atleast the memory B cell repertoire (2) generate a positional weightmatrix (PWM) of amino acid positional variability in the CDR1 and CDR2of the non-human species and the human species by calculating therelative frequency of amino acids at each position of a specificnon-human V-gene and each member of the subset of preferred acceptorscaffolds from humans; (3) blend each of two PWMs from the non-humanV-gene and a V-gene from the human preferred acceptor scaffolds toproduce a hybrid PWM that contained the amino acid variation observedfrom both species at each position; (4) optionally adjust the blendedPWM to remove cysteine, methionine, and tryptophan residues in order togenerate superior therapeutics; and (5) optionally adjust thecomposition of the blended PWM to bias the library more towards eitherthe human or non-human molecules, as well as to adjust the effectivediversity of the library.

The PWMs can be converted to instructions to produce a library ofnucleic acid molecules encoding immunoglobulin molecules. Nucleic acidsynthesis instructions are generated such that each amino acid isencoded at a position at approximately its frequency within the PWM,with the frequency of each amino acid position independent of theothers.

The in silico nucleic acid library produced by these methods can beanalyzed by probabilistic simulation to analyze the predicted propertiesof the synthesized library. The expected frequency of every molecule inthe library can be calculated. Mathematical simulation can be used toexplore the theoretical humanization of non-human antibodies to evaluatethe proximity of the closest humanizations, and the blended PWM can beadjusted to bias the libraries towards a given level of humanization.This is accomplished by iterative Monte Carlo sampling or other methods.

The methods can be used to ensure that for any given epitope of 5-25amino acids, the library will highly preferentially explore solutions nomore than 21 amino acids away from the non-human repertoire and no morethan 2 amino acids away from the human repertoire for the epitope inquestion.

The PWM can then be converted into synthesis instructions to produce asynthetic library, where each position in the CDRs1 and CDR2 contain thediversity observed in the blended library, and the frameworks are human.

The library can be assembled taking CDR-H3s and optimally CDR-L3s from anon-human species post-immunization or post-immune mediated events andtransferred into the library by restriction, Gibson overlap extension,PCR overlap extension, or other technologies.

The humanized immunoglobulins can be characterized by their bindingaffinity to the antigen and proteins with high amino acid similarity tothe antigen, such as the homologous protein in a related species. Thehumanized antibodies can be characterized to identify those that lackbinding affinity for off-target molecules. The human antibodies can becharacterized to determine their stability and aggregationkinetics/thermodynamics. The population of nucleic acids encoding theantibodies having the desired characteristics can be sequenced, andamino acid residues that are present at higher frequencies in lesssuccessful antibodies (e.g. antibodies with binding affinity below adesired level of affinity, such as an affinity level described herein)or amino acid residues that are present at higher frequencies in moresuccessful antibodies (e.g. antibodies with binding affinity above adesired threshold, such as an affinity level described herein) can bedetermined. Step (j) of antibody optimization can be accomplished byusing the characteristics of unsuccessful and successful antibodies tosynthesize novel antibodies that lack particular amino sequenceassociated with less successful antibodies and possess particular aminoacid sequence associated with more successful antibodies, therebyoptimizing antibodies.

Example 2. Determination of Appropriate Receptor Frameworks for MurineAntibodies and Characterization of their Frequencies

Germline V gene segments from mice can be compared to germline V genesegments from humans to identify preferred acceptor frameworks. Multiplegermline mouse V gene segments can be mapped onto individual germlinehuman V gene segments, thereby funneling much of the mouseimmunoglobulin repertoire towards particular preferred acceptorframeworks.

RNA from murine lymphocytes can be isolated and the sequences of theimmunoglobulins genes determined. The frequencies of the differentlight-chain immunoglobulin and heavy-chain immunoglobulin V genesegments that map to each human germline V gene segment can bedetermined. For example, multiple mouse V gene segments that map ontothe human acceptor V gene segment IGHV1-3 can be found in the sequencesassayed. An exemplary PWM for human immunoglobulin IGHV1-3 CDR sequencesis illustrated in FIG. 3.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of producing a population of nucleicacids encoding at least one protein comprising at least oneimmunoglobulin variable domain having a non-human-derived CDR3 aminoacid sequence embedded in essentially human framework sequences, whereinthe method comprises the following steps: (a) providing at least onenucleic acid encoding a non-human-derived complementarity determiningregion 3 (CDR3) amino acid sequence or an amino acid sequence furtherencompassing 1, 2, or 3 amino acids N-terminal and/or C-terminal of thenon-human-derived CDR3 amino acid sequence, (b) generating a populationof nucleic acids encoding at least one protein comprising at least oneimmunoglobulin variable domain having a non-human CDR3 amino acidsequence of step (a) embedded in essentially human framework sequences,wherein the human framework sequences comprise a first human frameworkregion (FR1), a second human framework region (FR2), a third humanframework region (FR3), and a fourth human framework region (FR4), suchthat the FR1 and FR2 regions are interspaced by a complementaritydetermining region 1 (CDR1), the FR2 and FR3 regions are interspaced bya complementarity determining region 2 (CDR2), and the FR3 and FR4regions are interspaced by a non-human-derived CDR3 amino acid sequence,wherein the nucleic acid sequences encoding the CDR1 and CDR2 amino acidsequences are diversified among the population of nucleic acids encodingat least one protein comprising at least one immunoglobulin variabledomain, wherein each nucleic acid sequence encoding a CDR1 or CDR2 aminoacid sequence is independently based (i) on a nucleic acid sequenceencoding a human CDR1 or CDR2, respectively, or (ii) on a nucleic acidsequence encoding a non-human CDR1 or CDR2, respectively, wherein atleast some of the nucleic acid sequences encoding a CDR1 or CDR2 aminoacid sequence have been modified to encode at least one amino acidpresent in non-human CDR1 or CDR2 amino acid sequences, respectively, incase of human CDR1 or CDR2, respectively, or to encode at least oneamino acid present in human CDR1 or CDR2 amino acid sequences,respectively, in case of non-human CDR1 or CDR2, respectively, andwherein the human FR1, FR2, FR3 and FR4 regions are human frameworkregions selected to provide a scaffold conducive for non-human CDR3amino acid sequences, with the proviso: that the two C-terminal aminoacids of FR2 are optionally non-human, and that the two C-terminal aminoacids of FR3 are optionally non-human.
 2. A method of producing apopulation of nucleic acid sequences encoding at least one proteincomprising at least one immunoglobulin variable domain having anon-human-derived CDR3 amino acid sequence embedded in essentially humanframework sequences, the method comprising one or more steps of aprocess as described herein.
 3. A method of producing a population ofamino acid sequences for one or more proteins comprising at least oneimmunoglobulin variable domain having a non-human-derived CDR3 aminoacid sequence embedded in essentially human framework sequences, themethod comprising one or more steps of a process as described herein. 4.A population of nucleic acids in accordance with claim
 1. 5. Apopulation of proteins in accordance with claim
 1. 6. A system forperforming a method of claim 1, the system comprising a computerprocessor programmed to perform the steps of the method.
 7. A computerreadable medium comprising machine executable code that upon executionby one or more computer processors implements a method of claim
 1. 8.Use of a composition in accordance with claim 1 in the preparation for amedicament for the treatment of a disease or condition of a subject. 9.A method of treating a subject for a disease or condition, comprisingadministering to a subject one or more proteins of claim
 1. 10. Anantibody library comprising a plurality of 10³ or more differenthumanized antibodies, wherein each humanized antibody of the pluralityof humanized antibodies comprises an immunoglobulin variable domainpolypeptide comprising, in an N-terminal to C-terminal direction, ahuman first framework region (FR1), a blended complementaritydetermining region 1 (CDR1), wherein each amino acid position of theblended CDR1 comprises an amino acid residue from either a human speciesor a non-human species, a human second framework region (FR2), a blendedcomplementarity determining region 2 (CDR2), wherein each amino acidposition of the blended CDR2 comprises an amino acid residue from eithera human species or a non-human species, a human third framework region(FR3), a non-human complementarity determining region 3 (CDR3), and ahuman fourth framework region (FR4); and wherein for the plurality ofhumanized antibodies: a) the CDR1s and the CDR2s comprise a plurality ofsequences; b) the CDR1s, at each amino acid position, comprise an aminoacid residue at a relative frequency within the antibody library that isa blend of: a relative frequency at the amino acid position in naturallyoccurring, human CDR1s, and a relative frequency at the amino acidposition in naturally occurring, non-human CDR1s; and c) the CDR2s, ateach amino acid position, comprise an amino acid residue at a relativefrequency within the antibody library that is a blend of: a relativefrequency at the amino acid position in naturally occurring, humanCDR2s, and a relative frequency at the amino acid position in naturallyoccurring, non-human CDR2s.
 11. The library of claim 10, wherein eachamino acid sequence of the CDR1 comprises at least one amino acid from anon-human CDR1 amino acid sequence and at least one amino acid from ahuman CDR1 amino acid sequence, and each amino acid sequence of the CDR2comprises at least one amino acid from a non-human CDR2 amino acidsequence and at least one amino acid from a human CDR2 amino acidsequence.
 12. The library of claim 10, wherein each amino acid sequenceof the CDR1 or the CDR2 exhibits at least 30% sequence identity to anon-human CDR1 or a non-human CDR2 sequence, respectively.
 13. Thelibrary of claim 10, wherein each amino acid sequence of the CDR1 or theCDR2 exhibits at least 30% sequence identity to a human CDR1 or a humanCDR2, respectively.
 16. The library of claim 10, wherein a similaritybetween a CDR1's or a CDR2's immunoglobulin variable domain and a CDR3'snative immunoglobulin variable domain is increased with respect to afeature selected from the group consisting of amino acid sequence,cosmology, length, canonical structure, heavy/light interface mountangle, and any combination thereof.
 17. The library of claim 10, whereintwo C-terminal amino acids of the FR2 are from non-human sequences. 18.The library of claim 10, wherein two C-terminal amino acids of the FR3are from non-human sequences.
 19. The library of claim 10, wherein eachhumanized antibody comprises a peptide that allows display of ahumanized antibody on a virus, a cell, or a surface.
 20. The library ofclaim 19, wherein the peptide is A-agglutinin-binding subunit (Aga2p).21. The library of claim 19, wherein the peptide is protein III (pIII).22. The library of claim 10, wherein each humanized antibody of theplurality of humanized antibodies comprises a scFv, a Fab, a Fab′,F(ab′)₂, or a Fv.
 23. The library of claim 10, wherein the sequence ofthe CDR1 or the sequence of the CDR2 does not comprise cysteine,methionine, and tryptophan residues.